Targets of activity

The murine transcription factor lymphocyte enhancer binding factor 1 (LEF-1) recognizes a minimal wingless response sequence in the midgut enhancer of Ultrabithorax. This visceral mesoderm enhancer, located 2.9 kb from the Ubx start site contains adjacent elements that respond to wg and dpp signaling. The DPP response sequence within this enhancer is a cAMP-response element (CRE). Wingless and DPP act independently but synergistically through this enhancer to stimulate Ubx expression in the midgut. The LEF-1-binding site contains an excellent match to the LEF-1 binding site first identified in the T cell receptor alpha chain enhancer. LEF-1 binds the Ubx wingless response sequence (WRS) with high affinity and specificity (Riese, 1997).

Decapentaplegic is an extracellular signal of the transforming growth factor-ß family with multiple functions during Drosophila development. For example, it plays a key role in the embryo during endoderm induction. During this process, Dpp stimulates transcription of the homeotic genes Ultrabithorax in the visceral mesoderm and labial in the subjacent endoderm. A cAMP response element (CRE) from an Ultrabithorax enhancer mediates Dpp-responsive transcription in the embryonic midgut, and endoderm expression from a labial enhancer depends on multiple CREs. The enhancer, called Ubx B confers Wingless- and Decapentaplegic-dependent expression in the visceral mesoderm. The Drosophila CRE-binding protein dCREB-2 binds to the Ultrabithorax CRE. Other transcription factors act through the Ubx B enhancer to confer its tissue-specific response to Dpp in the visceral mesoderm. CRE needs to cooperate with an LEF-1 binding site to respond to the Dpp signal in the visceral mesoderm. Adjacent to the CRE is another palindromic sequence that antagonizes the activating effects of Dpp and Wg signaling on the Ubx B enhancer. Therefore, a dCREB-2 protein may act as a nuclear target, or as a partner of a nuclear target, for Dpp signaling in the embryonic midgut (Eresh, 1997).

Mouse LEF-1 was used in these experiments because the endogenous protein (now known to be Pangolin) had not yet been identified. The WRS is recognized by LEF-1 in a ternary complex with Armadillo protein. Expressing LEF-1 throughout the mesoderm results in an anterior expansion of Ubx expression in the visceral mesoderm. A similar anterior expansion is observed after the expression of arm throughout the visceral mesoderm. Under these circumstances the second midgut constriction appears precociously and tends to form as a double constriction. LEF-1 activity depends on arm, since LEF-1 fails to stimulate Ubx transcription in arm mutants. In contrast, LEF-1 expressing wg mutants show a moderate level of Ubx transcription in LEF-1 expressing embryos. This implies that LEF-1, perhaps by virtue of being overexpressed, bypasses the need for Wingless stimulation (Riese, 1997).

If overexpressing mouse LEF-1 functions independently of wingless signaling, why are its effects localized to certain regions of the midgut? One possibility is that LEF-1 activity may be restricted by the dpp signal, which itself is localized to the middle midgut region where most LEF-1 activity is seen. When LEF-1 and DPP are coexpressed in the mesoderm, UBX staining stretches through the visceral mesoderm, whereas the effects of LEF-1 or of DPP by themselves are regionally restricted. There is, in fact very little effect of mesodermally expressed LEF-1 in dpp mutants embryos (Riese, 1997).

LEF-1 ovexpression has phenotypic effects in other developmental contexts in which WG and DPP operate. LEF-1 was expressed in the wing disc. LEF-1 overexpression leads to many extra bristles on the notum, a derivative of the wing disc. This LEF-1 effect resembles the phenotype of shaggy mutant clones. The wings of these flies look highly abnormal. In strong LEF-1 transformants, the wings are rudimentary, but they frequently show tufts of bristles as observed in shaggy mutant clones. These bristle tufts are localized to the tip of the wing. The effects of LEF-1 in these wings mimic loss of wingless function, such as missing wing margin bristles and gaps in the wing margin itself. Apparently, mouse LEF-1 overexpression in the margin primordium somehow interferes with the function of the endogenous wingless target transcription factor in a dominant negative way (Riese, 1997).

The requirement for both wingless and dpp signaling in the midgut is a prime example of how two genes acting together can result in a highly localized emergent structure. Clearly, the signaling inputs from wg and dpp are independent and separate until they reach their final target, the Ubx enhancer, where they converge at neighboring response elements. Neither signal response element on its own is sufficient, to stimulate transcription in the visceral mesoderm (Riese, 1997).

The optimal DNA-binding site was determined using a PCR-based binding site selection technique. The consensus sequence is CCTTTGATCTT, matching well with the canonical TCF binding motif CCTTTGA/TA/T (van de Wetering, 1997).

In wingless mutants, engrailed expression comes on normally but fails to be maintained. In a null pangolin mutant, engrailed expression is initiated normally, but the stripes of engrailed expression begin to decay by late stage 9, particularly in midlateral regions and along the ventral midline. This effect resembles that of a zygotic armadillo mutation or of removal of functional Wingless at the end of stage 9 (van de Wetering, 1997).

Mutations in the wingless pathway affect the expression of Ultrabithorax in the visceral mesoderm, disrupting the secondary midgut constriction. Indeed, Ultrabithorax expression is not maintained in pangolin mutants, while the secondary midgut constriction is absent. The primary constriction is not affected and does not move posteriorly. This contrasts with other mutations that disrupt the secondary midgut constriction, indicating that the Ubx regulatory network might be only partially disruption by pangolin mutation (van de Wetering, 1997).

Ras pathway specificity is determined by the integration of multiple signal-activated and tissue-restricted transcription factors

Ras signaling elicits diverse outputs, yet how Ras specificity is generated remains incompletely understood. Wingless and Decapentaplegic confer competence for receptor tyrosine kinase-mediated induction of a subset of Drosophila muscle and cardiac progenitors by acting both upstream of and in parallel to Ras. In addition to regulating the expression of proximal Ras pathway components, Wg and Dpp coordinate the direct effects of three signal-activated transcription factors (Pangolin/dTCF, Mad, and Pointed that function in the Wg, Dpp, and Ras/MAPK pathways, respectively) and two tissue-restricted transcription factors (Twist and Tinman) on a progenitor identity gene enhancer. The integration of Pointed with the combinatorial effects of dTCF, Mad, Twist, and Tinman determines inductive Ras signaling specificity in muscle and heart development (Halfon, 2000).

Cell fate specification in the somatic mesoderm of the Drosophila embryo has been examined as a model for dissecting the molecular basis of combinatorial signaling involving receptor tyrosine kinases (RTKs). The somatic musculature and the cells that compose the heart develop from specialized cells called progenitors. Each progenitor divides asymmetrically to produce two founder cells that possess information that specifies individual muscle fate and that seed the formation of multinucleate myofibers. The focus of this study has been a small subset of somatic mesodermal cells that express the transcription factor Even skipped. Eve is expressed in the progenitors and founders of both the dorsal muscle fiber DA1 and a pair of heart accessory cells, the Eve pericardial cells or EPCs. Since eve is the earliest known marker for these cells and is required for their formation, eve is referred to here as a progenitor identity gene (Halfon, 2000).

Previous genetic experiments have defined multiple intercellular signaling events that govern the progressive determination of the Eve progenitors. Signaling from both the Wnt family member Wingless (Wg) and the TGF family member Decapentaplegic (Dpp) prepatterns the mesoderm and renders cells competent to respond to Ras/MAPK activation. Localized Ras activation within the competence domain determined by the intersection of Wg and Dpp expression occurs through the action of two RTKs: the Drosophila epidermal growth factor receptor (Egfr) and the Heartless (Htl) fibroblast growth factor receptor. This RTK signaling induces two distinct equivalence groups, each of which expresses Eve. Lateral inhibition mediated by Notch then selects a single progenitor from each equivalence group (Halfon, 2000).

The present study explores how the prepattern genes wg and dpp establish competence for mesodermal cells both to activate and to respond to the Ras/MAPK cascade; how multiple intercellular signals are integrated to establish Eve progenitor fates, and how muscle- and cardiac-specific responses to Ras signaling are generated. Wg provides competence for the generation of the Ras/MAPK inductive signal by regulating the expression of key proximal components of the Egfr and Htl RTK pathways. Wg and Dpp then create competence for a specific response to the inductive signal both through their own respective downstream transcriptional effectors, dTCF and Mothers against dpp (Mad), and through their regulation of the mesoderm-specific transcription factors Tinman (Tin) and Twist (Twi). Specificity of the Ras/MAPK response is achieved though the integration of these signal-activated and tissue-restricted transcription factors, along with the Ras/MAPK-activated Ets domain transcription factor PointedP2 (Pnt), at a single transcriptional enhancer. These results provide a direct link between the initial axis patterning processes in the early embryo and the subsequent combinatorial signaling events that lead to the progressive determination of muscle and cardiac progenitors (Halfon, 2000).

The Eve progenitors in each mesodermal hemisegment arise during embryonic stage 11 in a dorsal region demarcated by the intersecting domains of Wg and Dpp expression. The cells exposed to both Wg and Dpp are competent to respond to localized Ras signaling, which induces the initial expression of Eve in two clusters of equipotent cells. In each of these equivalence groups, activity of the Notch pathway leads to the rapid refinement of Eve expression to a single muscle or cardiac progenitor. The two Eve equivalence groups arise sequentially. Cluster C2, from which progenitor P2 derives, is first to form. P2 divides asymmetrically, with one daughter maintaining Eve expression and becoming the founder of the two EPCs (F2EPC), and the other losing Eve expression and becoming the founder of muscle DO2. The second Eve-expressing cluster, C15, forms slightly later and produces the progenitor P15, which in turn divides to yield the founder of the Eve-expressing muscle, DA1, and an Eve-negative cell of as-yet-undetermined identity. Activation of the Ras/MAPK pathway in C15 depends on both the Egfr and Htl RTKs, but only Htl signaling is required for C2 formation (Halfon, 2000).

The progressive determination of Eve mesodermal progenitors requires that Wg prepattern the mesoderm, rendering cells competent to respond to inductive RTK/Ras signaling. To further investigate the basis of this competence, whether or not the Ras pathway is active in the absence of Wg signaling was examined by monitoring the expression of the activated, diphosphorylated form of MAPK in wg mutant embryos. Diphospho-MAPK is expressed in progenitor P2 in early stage 11 wild-type embryos. Not only is this progenitor missing from wg mutant embryos, but activation of MAPK in the C2 equivalence group, which is dependent on Htl, fails to occur. Similarly, Wg is essential both for P15 formation and for the Egfr- and Htl-dependent activation of MAPK in the equivalence group from which this progenitor is derived (Halfon, 2000).

Next to be determined was at what level in the RTK/Ras pathway Wg is required for MAPK activation. In wg mutant embryos, there is loss of (1) the P2-specific expression of Htl; (2) its specific downstream signaling component, Heartbroken (Hbr, also known as Dof and Stumps), and (3) Rhomboid (Rho), a protein involved in the presentation of the Egfr ligand Spitz. Conversely, constitutive Wg signaling, achieved by ectopic expression of Wg or an activated form of the downstream Wg pathway component Armadillo (Arm), induces Htl, Hbr, and Rho expression in more dorsal mesodermal cells than the single P2 progenitor found at a comparable developmental stage. This effect is less prominent for Rho than for Htl and Hbr, which may reflect different threshold responses to Wg. Alternatively, the effect on Htl and Hbr may be more pronounced because ectopic Wg signaling prolongs their earlier expression in the entire C2 cluster; Rho, in contrast, is normally expressed in P2 but not in C2, possibly making it more refractory to a prepattern factor such as Wg. Expanded expression of these RTK pathway components is associated with increased MAPK activation and Eve expression. However, these effects of Wg hyperactivation are transient, with a normal number of Eve progenitors eventually segregating. Moreover, activated Arm is able to fully rescue Htl, Hbr, Rho, diphospho-MAPK, and Eve expression in wg mutant embryos. Htl, Hbr, and Rho expression, as well as MAPK activation, are also Dpp dependent. In summary, Wg and Dpp regulate the production of several key proximal components of the Egfr and Htl signal transduction pathways (Halfon, 2000).

Given the involvement of Wg in the expression of Htl, Hbr, and Rho, it was reasoned that a constitutively activated form of Ras1 might bypass the requirement of Wg for MAPK activation. Constitutively activated Ras1, when targeted to the mesoderm of wild-type embryos, leads to an overproduction of Eve progenitors, as well as to the expected hyperactivation of MAPK in these cells. In the absence of Wg signaling, diphospho-MAPK expression is restored by activated Ras1. However, despite this recovery of MAPK activation, constitutive Ras1 does not rescue Eve progenitor formation in a wg mutant background. This is in marked contrast to the ability of activated Arm to fully rescue RTK signaling and Eve progenitor specification in a wg mutant. These results suggest that, in addition to enabling activation of Ras/MAPK signaling as a result of the induction of Htl, Hbr, and Rho expression, Wg signaling must contribute other factors that are essential for the specification of mesodermal Eve progenitors (Halfon, 2000).

One mechanism that would ensure the convergence of the multiple regulatory inputs required for the formation of P2 and P15 is integration by a transcriptional enhancer. Since Eve expression is the feature that uniquely identifies these progenitors, an investigation was made of whether eve itself is a direct target for regulation by both signal-activated and tissue-specific transcription factors. Regulatory sequences responsible for mesodermal eve expression are located approximately 6 kb downstream of the transcription start site. Deletions of this region were generated and a 312 bp minimal enhancer was defined that has been termed the eve Muscle and Heart Enhancer (MHE). When fused to a nuclear-lacZ reporter gene, the MHE drives expression in a mesodermal pattern identical to that of the endogenous eve gene. Reporter expression initiates at early stage 11, coincident with the onset of Eve expression in the equivalence group C2. Following formation of P2, MHE activity is observed in P15 and in the P2 daughters, F2EPC and F2DO2, then in the EPCs and the F15 daughters of P15, and finally in muscle fiber DA1. Colocalization of MHE-driven ß-galactosidase expression with Runt, which marks the F2DO2 founder and muscle DO2, establishes that the reporter gene expression present in Eve-negative sibling cells is a result of ß-galactosidase perdurance. Of note, the MHE mimics endogenous Eve expression despite its lack of a consensus binding site for the transcription factor Zfh-1 that had previously been proposed to play a role in mesodermal eve regulation (Halfon, 2000).

Strikingly, the MHE is only active in a single nucleus of the mature DA1 and DO2 muscles. It is inferred that these are the original nuclei of the F15DA1 and F2DO2 founders based on prior reporter expression in those cells. Similar results were obtained when DNA flanking the MHE by several hundred base pairs on either side (+4.96 to +7.36 kb), including the previously described Zfh-1 site, was included in the reporter construct, or when the MHE was placed 3' to a reporter gene fused to the endogenous eve promoter. Thus, additional sequences are required for eve expression in non-founder myofiber nuclei. Of critical importance to the present study, the MHE fully recapitulates mesodermal Eve expression during the signal-dependent induction of progenitor and founder cells (Halfon, 2000).

Genetic manipulation of the Wg, Dpp, and RTK/Ras signaling pathways causes predictable alterations of endogenous mesodermal Eve expression. A determination was made of whether the isolated MHE responds appropriately to these signals. In all genetic backgrounds, reporter gene expression corresponds precisely to that of endogenous eve. For example, constitutively activated Arm transiently increases the expression of both genes. However, Wg hyperactivation does not have a stable effect on MHE function. In contrast, both endogenous eve and the MHE-driven reporter are induced throughout the initial competence domain by constitutively activated Pnt, and expression of both markers extends laterally in the presence of activated Arm plus Pnt. Ectopic Dpp leads to both endogenous Eve and MHE-driven reporter expression in the ventral mesoderm, while coexpression of Dpp and activated Ras1 induces expression of both genes in a dorsal-ventral stripe. These results demonstrate that the isolated MHE is responsive to all of the known signals that are essential for the specification of Eve progenitors (Halfon, 2000).

Given that the MHE recapitulates early mesodermal Eve expression, a determination was made of whether this enhancer contains binding sites for candidate signal-dependent and mesoderm-specific transcription factors. Focus was placed on two mesoderm-specific factors, Tin and Twi, as well as the nuclear factors that act downstream of Wg (dTCF), Dpp (Mad) and Ras (Pnt, Yan). A computer-based search of the MHE sequence has suggested the presence of potential binding sites for each of these transcription factors. Gel-shift assays confirm that these putative sites actually bind the relevant factors. This analysis establishes the existence of one binding site for dTCF, six for Mad, two for Twist, and four each for Tin and Pnt. Since Yan binds to each of the Pnt sites, these are referred to as Ets sites (Halfon, 2000).

To ascertain whether these in vitro binding sites have in vivo functional significance, the sites were mutated, both singly and in combination, within the context of the entire MHE. All mutagenesis was by base substitution so as not to affect the spacing between other potential cis-regulatory elements. The ability of the mutated MHEs to drive reporter gene expression was tested in transgenic embryos and this expression was compared to that of endogenous Eve. Of the six Mad sites, only Mad4, 5, and 6 are critical for MHE function when inactivated singly or in combination. Mutation of the single dTCF site or of individual binding sites for Twi, Tin, or the Ets factors also lead to loss of reporter gene expression in some, but not all, Eve-expressing cells, with some mutant sites associated with a more severe loss than others. Of note, both the EPC and DA1 lineages are affected equally by all of the mutations. In addition, the activity level in those Eve-expressing cells that do maintain reporter gene expression is on average lower than that seen with the wild-type MHE. In contrast to the single site mutants, mutation of the two Twi, all four Tin, or all four Ets sites completely eliminate MHE activity. It is concluded that binding sites for two tissue-specific and three signal-responsive transcription factors are required for full activity of the MHE in both the muscle and the heart lineages (Halfon, 2000).

The finding that the three Wg-dependent factors, dTCF, Twi, and Tin, that directly regulate eve could explain why activated Ras is incapable of bypassing Wg in the induction of Eve progenitors. Therefore attempts were made to rescue Eve expression in wg mutant embryos by ectopically expressing Twi and Tin together with activated Ras. However, Eve progenitors were not recovered by this manipulation, perhaps due to the direct requirement of dTCF for eve MHE activity. While activated Arm can supply the missing downstream Wg transcription factor in this rescue experiment, Arm alone is capable of fully rescuing not only the Eve progenitors but also all of the Wg-dependent factors that regulate the MHE, including Twi, Tin, and the RTK/Ras pathway components. Thus, the combined effects of the MHE transcription factors could not be further evaluated in the absence of Wg signaling. Nevertheless, the rescue and enhancer mutagenesis data strongly support the involvement of Wg as a mesodermal competence determinant both upstream of the Ras pathway and directly (via dTCF) as well as indirectly (via Twi and Tin) in the transcriptional response to inductive RTK signaling (Halfon, 2000).

Since mutation of any single transcription factor binding site in the MHE causes only a partial loss of enhancer activity, it was considered whether different sites might function together synergistically. To test this possibility, binding site mutations for two different activators were combined. Simultaneous mutation of the dTCF and Twi1 sites led to reporter gene expression in approximately 5-fold fewer cells than would be expected from the additive independent effects of each mutation. A similar, though slightly less robust, synergy was observed when the dTCF and Ets3 mutations were combined (Halfon, 2000).

An assessment was made of whether ectopic coexpression of individual transcription factors or upstream signals would lead to cooperative effects on endogenous Eve expression. As previously reported, ectopic Wg has no effect on Eve expression at late stage 11, activated Ras1 induces extra Eve progenitors, and ectopic Wg plus activated Ras1 cause a lateral expansion of the progenitor clusters. When Twi is expressed using a twi-Gal4 driver, a few Eve-positive cells develop at ectopic positions. The magnitude of this effect is increased by coexpression of Wg and Twi, and even more so by coexpression of Twi with activated Ras1. The latter effect strikingly resembles that of Wg plus activated Ras1. With the simultaneous ectopic expression of Wg, Twi, and activated Ras1, Eve progenitors form an almost continuous anteroposterior stripe confined to the dorsal mesoderm. These results demonstrate a synergistic induction of Eve progenitors by various combinations of Wg, Twi, and activated Ras1 that parallels the synergistic loss of MHE activity seen by mutating the dTCF, Twi, and Ets binding sites. Taken together, these loss- and gain-of-function findings suggest that dTCF, Twi, and Pnt cooperate at the MHE to synergistically regulate Eve transcription and, by extension, to induce the specification of Eve progenitor fates (Halfon, 2000).

It is concluded that Wg and Dpp coordinate a series of signal-activated (dTCF and Mad) and mesoderm-specific (Twi and Tin) transcription factors in a temporal and spatial pattern that facilitates cooperation with the Ras transcriptional effector Pnt. The synergistic integration of these five transcription factors by a single enhancer generates a specific developmental response to Ras/MAPK signaling. Moreover, Wg and Dpp exert proximal effects in this signaling network by enabling Ras/MAPK activation through the regulated localized expression of upstream components of the RTK signal transduction machinery. A model governing the acquisition of developmental competence, signal integration and response specificity in this system is presented. Wg and Dpp provide competence through the regulation of tissue-specific transcription factors (Tin and Twi), signal-responsive transcription factors (Mad and dTCF), and proximal components of the RTK/Ras pathways (Htl, Hbr, and Rho). The Ras signaling cascade leads to activation of the inductive transcription factor, Pnt, and inactivation of the Yan repressor. While a direct role for Mad in regulating Tin expression has been demonstrated, Wg regulation of Tin, Twi, Htl, Hbr, and Rho may be either direct or indirect. Dpp has additional effects on the proximal RTK factors. The five transcriptional activators assemble at and are integrated by the MHE, where they function synergistically to promote eve expression. Specificity of the response to inductive RTK/Ras signaling derives from the combinatorial effects of the tissue-restricted and signal-activated transcription factors that converge at the MHE. In the absence of inductive signaling, Yan would repress eve by binding to the Ets sites. Since eve is a muscle and heart identity gene, the regulatory mechanisms are inferred to have a more general function in determining progenitor fates. Additional complexity attendant upon the control of RTK activity in this system derives from positive feedback regulation of the Ras/MAPK cascade and from reciprocal regulatory interactions between the Ras and Notch pathways (Halfon, 2000).

Molecular integration of inductive and mesoderm-intrinsic inputs governs even-skipped enhancer activity in a subset of pericardial and dorsal muscle progenitors

Individual somatic muscles and heart progenitors are specified at defined positions within the mesodermal layer of Drosophila. The expression of the homeobox gene even-skipped (eve) identifies one specific subset of cells in the dorsal mesoderm, which give rise to particular pericardial cells and dorsal body wall muscles. Genetic analysis has shown that the induction of eve in these cells involves the combined activities of genes encoding mesoderm-intrinsic factors, such as Tinman (Tin), and spatially restricted signaling activities that are largely derived from the ectoderm, particularly those encoded by wingless and decapentaplegic. A Dpp-activated Smad protein, phosphorylated Mad, is colocalized in eve-expressing cells during an extended developmental period. A mesodermally active enhancer of eve contains several Smad and Tin binding sites that are essential for enhancer activity in vivo. This enhancer also contains a number of binding sites for the Wg-effector Pangolin (Pan/Lef-1), that are required for full levels of enhancer activity. However, the main function of these sites is to prevent ectopic enhancer activity in the dorsal mesoderm. This suggests that, in the absence of Wg signaling, Pan binding serves to abrogate the synergistic activities of Smads and Tin in eve activation, while in cells that receive Wg signals, Pan is converted into a coactivator that promotes eve induction. Together, these data show that the eve enhancer integrates several regulatory pathways via the combinatorial binding of the mesoderm-intrinsic regulator Tin and the effectors of the Dpp and Wg signals (Knirr, 2001).

Cubitus interruptus is necessary but not sufficient for direct activation of a wing-specific decapentaplegic enhancer

In Drosophila, the imaginal discs are the primordia for adult appendages. Their proper formation is dependent on the activation of the decapentaplegic gene in a stripe of cells just anterior to the compartment boundary. In imaginal discs, the dpp gene has been shown to be activated by Hedgehog signal transduction. However, an initial analysis of its enhancer region suggests that its regulation is complex and depends on additional factors. In order to understand how multiple factors regulate dpp expression, focus was placed on a single dpp enhancer element, the dpp heldout enhancer, from the 3' cis regulatory disc region of the dpp locus. A molecular analysis of this 358 bp wing- and haltere-specific dpp enhancer is presented that demonstrates a direct transcriptional requirement for the Cubitus interruptus (Ci) protein. The results suggest that, in addition to regulation by Ci, expression of the dpp heldout enhancer is spatially determined by Drosophila TCF (dTCF or Pangolin) and the Vestigial/Scalloped selector system and that temporal control is provided by dpp autoregulation. Consistent with the unexpectedly complex regulation of the dpp heldout enhancer, analysis of a Ci consensus site reporter construct suggests that Ci, a mediator of Hedgehog transcriptional activation, can only transactivate in concert with other factors (Hepker, 1999).

The dppho enhancer (so named because mutations in the region result in a 'held out' wing phenotype) was chosen for detailed analysis because this small region contains a cluster of putative transcription factor binding sites that is conserved in Drosophila virilis. The dppho enhancer is located from map position 111.9 to 112.3, approximately 18 kb from the 3' terminus of the dpp structural gene. The enhancer shares 52% sequence identity with the homologous region from D. virilis. Within the conserved sequences are found reasonable matches for the binding sites of several known transcription factors, including Engrailed, Ci, dTCF, Mothers against Decapentaplegic (Mad) and Scalloped. Of particular interest is the presence of potential Ci consensus binding sites. Gel mobility shift assays were performed with the DNA-binding domain of Ci and they demonstrated sequence-specific binding to the dppho fragment (Hepker, 1999).

The expression pattern of dppho-lacZ is consistent with this reporter being restricted by the extent of overlap between Hh and Wg signals in the wing pouch. For example, the dppho enhancer directs expression of lacZ reporter in a stripe coincident with high-level full-length Ci and endogenous dpp expression in the wing primordium of the wing imaginal disc. Furthermore, its expression is most robust in early larval stages and fades in a manner complementary to the dynamic pattern of wg expression in the wing disc. This enhancer also directs expression of a reporter ventrally in an analogous stripe in the haltere disc. Indeed ectopic expression data, together with clonal analysis, demonstrates that Ci and dTCF regulate dppho-lacZ expression, and this regulation is shown to be direct (Hepker, 1999).

Regulation of the dppho enhancer cannot be solely dependent on Wg and Hh signals since this element directs expression specifically in presumptive wing tissue. A candidate for a wing-specific factor involved in dppho regulation is the Vestigial/Scalloped transcriptional complex. The dppho sequence contains a weak match to the Sd/TEA DNA-binding site consensus, therefore a test was performed to see whether the Vg/Sd selector system is involved in restricting dppho-lacZ expression to the wing. A 30AGAL4 or an apterousGAL4 driver was used to direct expression of UAS-vg. In both cases, ectopic expression of vg induces expression of dppho-lacZ, but only near the A/P boundary. Similar experiments performed with UAS-sd result in loss of dppho-lacZ expression (Hepker, 1999).

decapentaplegic is a direct target of dTcf repression in the Drosophila visceral mesoderm

Drosophila T cell factor (dTcf: Pangolin) mediates transcriptional activation in the presence of Wingless signaling and repression in its absence. Wingless signaling is required for the correct expression of dpp in parasegments 3 and 7 of the Drosophila visceral mesoderm. A dpp enhancer element, which directs expression of a reporter gene in the visceral mesoderm in a pattern indistinguishable from dpp, has been shown to have two functional Pangolin binding sites. Mutations that reduce or eliminate Wingless signaling abolish dpp reporter gene expression in parasegment 3 and reduce it in parasegment 7 while ectopic expression of Wingless signaling components expand reporter gene expression anteriorly in the visceral mesoderm. However, mutation of the Pangolin binding sites in the dpp enhancer results in ectopic expression of reporter gene expression throughout the visceral mesoderm, with no diminution of expression in the endogenous sites of expression. These results demonstrate that the primary function of Pangolin binding to the dpp enhancer is repression throughout the visceral mesoderm and that activation by Wingless signaling is probably not mediated via these Pangolin binding sites to facilitate correct dpp expression in the visceral mesoderm (Yang, 2000).

Expression of dpp in the VM responds to Wg signaling. wg and pan mutations eliminate BE reporter gene expression in PS3 and slightly reduce it in PS7. A similar result has been reported for Dpp protein in an arm mutant background. These result is surprising because Drosophila embryos have a large amount of maternal embryonic PAN mRNA. It was not expected that the null pan phenotype would be similar to that of wg, which is expressed only zygotically. It has been concluded that Wg signaling is required for activation of dpp in PS3 and assists in the activation in PS7. It is also suggested that the maternal contribution of pan may not play a role in this activation. Ectopic expression of Wg or the constitutively active ArmS10 throughout the mesoderm results in the expansion of BE reporter gene expression from its endogenous site in PS7 through PS2. This expansion of expression is restricted to the VM with the exception of some faint staining in the presumptive somatic muscle precursors. A similar result was reported for Dpp protein in embryos mutant for sgg, which thus exhibit ectopic Wg signaling. Levels of reporter expression appear equally intense in all staining parasegments. Therefore, either PS3 expression must be intensified relative to PS7 expression or PS7 expression must be reduced relative to PS3, since BE expression in a wild-type background is significantly higher expression in PS7 than in PS3. Furthermore, other factors must be keeping dpp off, posterior to PS7. These include Abdominal-A, a homeodomain transcription factor that binds the same sites as Ubx and prevents dpp expression. While these results clearly demonstrate that Wg signaling results in activation of dpp in the VM, they are not yet conclusive as to whether this activation is indirect (Yang, 2000).

A search of the BE enhancer element reveals two sequences with a good match to the consensus Pan binding site. These dTcf sites might be used to directly activate dpp. However, mutation of both sites does not reduce expression directed by BE in either PS7 or PS3. Strikingly, BE reporter gene expression expands throughout the VM, even overcoming repression posterior to PS7. It had previously been reported that a fragment of BE between the BamHI and MscI restriction sites could direct reporter gene expression throughout the VM. This fragment lacks both Pan sites and all of the homeodomain binding sites. It is proposed that the two Pan binding sites in BE limit expression of this element by direct repression and that this could be the primary event regulated through these sites (Yang, 2000).

Expanded expression in the double pan site mutation approaches the levels seen in PS7 and PS3 but does not completely reach these levels. This is in contrast with ectopic Wg signaling where BE reporter gene expression from PS7 through PS3 is approximately the same intensity. It is concluded that there must be additional inputs to the BE fragment that further activate BE reporter gene transcription in PS7 and PS3, but that these are not directly regulated by Wg signaling through the Pan sites in BE. The most likely candidates for this activation function are: (1) Exd, which can function as a direct activator of the BE fragment in PS7 and PS3; (2) Ubx, which has already been demonstrated to directly bind the BE fragment and activate transcription, though this latter effect can only account for PS7 expression, and (3) Wg signaling to another unknown target gene, which in turn activates dpp. As noted above, Ubx itself has already been identified as a direct target of Pan activation. These results do not preclude a role for direct activation of Wg signaling on the wild-type BE enhancer through its Pan binding sites, both in reporter gene constructs and in the endogenous dpp gene. In other words, Wg signaling might still convert Pan from a repressor to an activator directly on the BE enhancer element; however, this effect is of less consequence in regulating the BE enhancer function than was initially predicted (Yang, 2000).

Mutation of either Pan binding site alone results in partial derepression of BE reporter gene expression. Derepression is much more pronounced when site T1 is mutated than when site T2 is mutated. The ectopic expression patterns of transgenic lines for the single-site mutations are weaker and more variable than either double-site mutation. This suggests that Pan proteins might be interacting synergistically on the two binding sites to repress dpp expression in the VM. In contrast to the results using the dpp enhancer, expression of Ubx in these same cells is mediated by activation through Pan sites (Yang, 2000).

There is an interesting dichotomy in these results. In one case, mutation of the Pan binding sites in BE results in derepression of reporter gene expression throughout the VM. In the other, embryos homozygous for a null pan mutation do not show derepression of a BE reporter gene. The large maternal contribution of wild-type Pan from heterozygous mothers might be sufficient to maintain the repressed state of a BE reporter gene throughout embryogenesis. However, pan and wg mutant embryos do not express the BE reporter gene in PS3 and show slightly reduced levels in PS7. In other words, with respect to activation of the BE reporter gene via Wg signaling, a null pan mutation is behaving like a complete block to Wg activation. Maternal Pan is not sufficient to substitute for zygotic gene product in Wg signaling activation of dpp. A model is proposed to explain these results. In early embryogenesis, maternal Pan binds BE and represses expression, by binding the corepressors Gro and CBP. Pan becomes modified, possibly by acetylation, and becomes refractory to conversion to an activator. There is no Wg signal and cytoplasmic Arm is phosphorylated by Sgg and degraded. Later in development, a general VM enhancer binding protein is predicted to be present, as the BamHI-MscI fragment of BE drives reporter gene expression throughout the VM and presumably dpp expression as well. In PS3 and PS7 of the VM, Wg signaling occurs, resulting in the stabilization of cytoplasmic Arm, which then combines with newly synthesized Pan (zygotic) and displaces maternal Pan to permit transcriptional activation in combination with the putative general VM enhancer binding protein, Exd, and Ubx (specifically in PS7). Outside of PS3 and PS7, zygotic Pan can gradually replace maternal Pan but, in the absence of stable Arm, it is rapidly converted to a repressor, which blocks function of the general VM enhancer. Finally, when the Pan binding sites are mutant, the general VM enhancer binding protein can constitutively activate BE reporter genes and this level of expression is increased in PS3 and PS7 by the binding of Exd and Ubx. Additional proteins may also play a role in this regulation. One prediction of this model is that removal of maternal Pan would result in derepression of the BE reporter gene throughout the VM (Yang, 2000).

The transcriptional repressor Brinker antagonizes Wingless signaling

In the embryonic midgut of Drosophila, Wingless (Wg) signaling elicits threshold-specific transcriptional response, that is, low-signaling levels activate target genes, whereas high-signaling levels repress them. Wg-mediated repression of the HOX gene Ultrabithorax (Ubx) is conferred by a response sequence within the Ubx B midgut enhancer, called WRS-R. It further depends on the Teashirt (Tsh) repressor, which acts through the WRS-R without binding to it. Wg-mediated repression of Ubx B depends on Brinker, which binds to the WRS-R. Brinker binds to a site distinct from that occupied by the Wg effector, the Pangolin/Armadillo activator complex. Brinker thus acts at short range to block the activity of this complex. Furthermore, Brinker blocks transcriptional activation by ubiquitous Wg signaling. Brinker binds to Tsh in vitro, recruits Tsh to the WRS-R, and mutual physical interactions are found between Brinker, Tsh, and the corepressor dCtBP. This suggests that the three proteins may form a ternary repressor complex at the WRS-R to quench the activity of the nearby-bound Pangolin/Armadillo transcription complex. Finally, brinker and tsh produce similar mutant phenotypes in the ventral epidermis, and double mutants mimic overactive Wg signaling in this tissue. This suggests that Brinker, which was initially discovered as an antagonist of Dpp signaling, may have a widespread function in antagonizing Wg signaling (Saller, 2002).

Most likely, Brinker uses a mechanism called quenching to block Pangolin/Armadillo. Quenching involves interaction of repressors (and the corepressors they recruit) with activators bound to nearby sites. Brinker is known to be able to quench target genes by recruiting the corepressor Groucho, which is involved in multiple quenching processes. groucho antagonizes wg, and TCF factors can bind to Groucho proteins directly, so Pangolin may thus be able to recruit Groucho unassisted. However, these findings do not rule out the possibility that Pangolin relies on cooperation with Brinker to achieve Groucho recruitment (Saller, 2002 and references therein).

The Drosophila midgut has provided a model system in which Wg signaling regulates gene transcription in a concentration-dependent manner; low signaling levels activate Wg target genes, whereas high levels repress the same genes. The discovery that Brinker confers transcriptional repression by Wg completes the picture of the DNA-binding proteins that interpret these different signaling thresholds. Pangolin confers Wg-induced stimulation of target genes, but its activity can be blocked by Brinker, which confers Wg-mediated repression of the same genes. Pangolin depends on Armadillo for its activity, whereas Brinker depends on Tsh to block the activity of the Pangolin/Armadillo complex. In turn, the availability of Armadillo depends directly on Wg signaling, which promotes its stabilization and nuclear translocation, whereas the availability of Tsh depends on transcription of its gene (which itself depends on wg). In other words, high Wg signaling induces locally the expression of the Tsh corepressor, which then cooperates with Brinker to repress Wg target genes in the same cells. One of these targets is wg itself, so Brinker and Tsh take part in the negative feedback loop of Wg signaling in the middle midgut (Saller, 2002).

Wingless effects mesoderm patterning and ectoderm segmentation events via induction of its downstream target sloppy paired

Inactivation of either the secreted protein Wingless (Wg) or the forkhead domain transcription factor Sloppy Paired (Slp) has been shown to produce similar effects in the developing Drosophila embryo. In the ectoderm, both gene products are required for the formation of the segmental portions marked by naked cuticle. In the mesoderm, Wg and Slp activities are crucial for the suppression of bagpipe (bap), and hence visceral mesoderm formation, and the promotion of somatic muscle and heart formation within the anterior portion of each parasegment. During these developmental processes, wg and slp act in a common pathway in which slp serves as a direct target of Wg signals that mediates Wg effects in both germ layers. Evidence has been found that the induction of slp by Wg involves binding of the Wg effector Pangolin (Drosophila Lef-1/TCF) to multiple binding sites within a Wg-responsive enhancer, located in 5' flanking regions of the slp1 gene. Based upon genetic and molecular analysis, it is concluded that Wg signaling induces striped expression of Slp in the mesoderm. Mesodermal Slp is then sufficient to abrogate the induction of bagpipe by Dpp/Tinman, which explains the periodic arrangement of trunk visceral mesoderm primordia in wild type embryos. Conversely, mesodermal Slp is positively required, although not sufficient, for the specification of somatic muscle and heart progenitors. It is proposed that Wg-induced slp provides striped mesodermal domains with the competence to respond to subsequent slp-independent Wg signals that induce somatic muscle and heart progenitors. It is also proposed that in wg-expressing ectodermal cells, slp is an integral component in an autocrine feedback loop of Wg signaling (Lee, 2000).

Combgap relays Wingless signal reception to the determination of cortical cell fate in the Drosophila visual system

The pattern of Combgap expression in the developing visual ganglia was examined by in situ hybridization and by staining with an anti-Cg antiserum. Both analyses gave similar results. Cg is expressed most strongly in dorsal and ventral regions of the optic ganglia, and weakly or not at all in the midline region. cg transcript and protein are reduced in the vicinity of the wg+ domains. The absence of Cg in the midline region is consistent with the lack of phenotypic effects of cg mutations in this region. Since ectopic wg+ activity is sufficient to induce the ectopic expression of wg target genes in the midline region, it is supposed that other factors are responsible for wg target gene regulation there. The reduction of cg expression found in the vicinity of wg+ target gene expression is consistent with the notion that wg+ induction of its optic lobe targets occurs via the downregulation of Cg expression (Song, 2000).

These observations along with the role of Cg as a negative regulator of wg target genes suggest that Cg expression might be regulated by wg+. Consistent with this notion, three consensus dTCF binding sites were identified within the first intron of the cg locus. To determine whether Cg expression is indeed under wg+ control, animals were generated carrying ectopic wg+ clones. The wg+ clones resulting from recombination between the repeated FRT sites were visualized by their failure to express the CD2 marker. The presence of ectopic wg+-expressing cells could also be inferred by the local nonautonomous induction of the target gene omb. The induction of omb by ectopic wg+ expression coincides with a reduction in Cg expression. This effect of wg+ is nonautonomous, as both the induction of Omb and the reduction of Cg expression have been found to extend beyond the boundary of marked wg+ clones. Cg expression thus appears to be under the nonautonomous control of wg+ activity (Song, 2000).

Drosophila segment borders result from unilateral repression of hedgehog activity by wingless signaling

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

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

stripe (sr) is expressed in all precursors of the epidermal muscle attachment sites, including those marking the segment border in the Drosophila larvae. To obtain an early molecular marker for the segment border corresponding to the row of cells posteriorly adjacent to the en expression domain, a 1.9 kb enhancer element of the sr gene (sr1.9) was isolated that is both necessary and sufficient to direct transgene-dependent lacZ expression in segment border precursor cells in a dorsal and lateral position of the embryo. Expression of the reporter gene is activated in parallel with sr, which is first expressed during late stage 10. At this time, the initial equal distribution of Wg has already become asymmetric, meaning that the protein spreads anteriorly over a range of maximally five cells but is restricted to only one row of cells directly adjoining the posterior margin of the expression domain. sr acts as a transcription factor required for setting up the cell fate of the muscle attachment sites which mark the segment border of the fly. Thus, sr1.9-dependent reporter gene expression can be employed to study the transregulatory requirement for positioning the segment border cells within the PS (Piepenburg, 2000).

To explore how Wg exerts its repressing function, whether the Drosophila TCF/Lef1 homolog Pangolin (Pan), the nuclear mediator of Wg activity, can directly interact with the sr239 element was examined. Pan in vitro binding sites were found next to and partially overlapping the Ci binding sites. Deletion of one Pan binding site that leaves the Ci binding sites intact (sr239DeltaPan), resulted in gene activation anterior to the en domain. In contrast to sr239-mediated gene expression that can be suppressed by ptc-Gal4-driven Wg activation, sr239?Pan-mediated gene expression is not abolished in response to ectopic Wg activity (Piepenburg, 2000).

It is known that Pan can associate with corepressors such as dCBP or Groucho. Upon reception of the Wg signal, Pan is switched into an activator of transcription by association with Armadillo, a coactivator of Wg target genes. The findings in this study suggest an alternative mechanism since the Pan binding sites of the sr1.9 and sr239 elements mediate Pan-dependent repression in cells with high Wg activity. This repression is necessary and sufficient to antagonize Ci-dependent transcriptional activation. Pan could thereby exert this function by competing sterically for Ci binding, by short-range quenching of Ci-mediated gene activation, or by active repression. Each way, Pan would allow for the formation of only one row of segment border cells within each PS by repressing the Hh-dependent sr activation in the wg domain (Piepenburg, 2000).

Pangolin represses Dpp

During germ band elongation, widespread dpp expression in the dorsal ectoderm patterns the underlying mesoderm. These Dpp signals specify cardial and pericardial cell fates in the developing heart. At maximum germ band extension, dpp dorsal ectoderm expression becomes restricted to the dorsal-most or leading edge cells (LE). A second round of Dpp signaling then specifies cell shape changes in ectodermal cells leading to dorsal closure. A third round of dpp dorsal ectoderm expression initiates during germ band retraction. This round of dpp expression is also restricted to LE cells but Dpp signaling specifies the repression of the transcription factor Zfh-1 in a subset of pericardial cells in the underlying mesoderm. Surprisingly, cis-regulatory sequences that activate the third round of dpp dorsal ectoderm expression are found in the dpp disc region. The activation of this round of dpp expression is dependent upon prior Dpp signals, the signal transducer Medea, and possibly release from dTCF-mediated repression. These results demonstrate that a second round of Dpp signaling from the dorsal ectoderm to the mesoderm is required to pattern the developing heart and that this round of dpp expression may be activated by combinatorial interactions between Dpp and Wingless (Johnson, 2003).

Genetic analysis demonstrates that some of the enhancers responsible for the second round of dpp LE expression are contained within the portion of BS3.21 that is not shared with BS3.22. An interspecific comparison shows that this region contains a stretch of 96 nucleotides that is 95% identical between two evolutionarily distant Drosophila species. An examination of this 96-base pair stretch reveals a candidate Mad/Med binding site that is 100% conserved between the species. This is consistent with results showing that BS3.21 expression requires dpp disc region sequences and that dpp151H expression requires Medea (Johnson, 2003).

Previous studies have shown that Wg signals repress dpp expression. One particularly relevant example is in the leg imaginal disc where Wg signaling represses dpp transcription within the wg expression domain. Two recent studies have been conducted to identify the factors and cis-regulatory sequences mediating Wg repression of dpp expression in leg discs. These studies show that either the expression of a dominant negative form of dTCF or the mutation of all dTCF binding sites in the reporter gene dpp-H3/Nhe eliminate Wg repression of dpp expression in leg discs. This suggests that Wg signaling represses dpp expression in leg discs via dTCF binding sites. These are the dTCF sites just outside of BS3.21 that are contained in all of the overlapping reporter genes that fail to display LE expression (Johnson, 2003).

A model is proposed for the regulation of the second round of dpp LE expression. In this model, combinatorial interactions between Dpp and Wg signals regulate the second round of dpp LE expression. It is proposed that during early stages of heart development Wg signals repress transcription from the disc region enhancers responsible for the second round of dpp LE expression via dTCF sites, just as Wg represses dpp expression in leg discs. Subsequently during germ band retraction, an as yet unidentified factor expressed in a subset of LE cells activates the second round of dpp LE expression. This factor appears to play a dual role, simultaneously disabling Wg repression and positively stimulating the second round of dpp LE expression. Once activated, Dpp autoregulation takes over to maintain the second round of dpp LE expression (Johnson, 2003).

Pangolin regulates a Serrate wing enhancer

Drosophila wing development is a useful model to study organogenesis, which requires the input of selector genes that specify the identity of various morphogenetic fields and cell signaling molecules. In order to understand how the integration of multiple signaling pathways and selector proteins can be achieved during wing development, the regulatory network that controls the expression of Serrate (Ser), a ligand for the Notch (N) signaling pathway, which is essential for the development of the Drosophila wing, as well as vertebrate limbs, was examined. A 794 bp cis-regulatory element located in the 3' region of the Ser gene can recapitulate the dynamic patterns of endogenous Ser expression during wing development. Using this enhancer element, Apterous (Ap, a selector protein), and the Notch and Wingless (Wg) signaling pathways, are shown to sequentially control wing development through direct regulation of Ser expression in early, mid and late third instar stages, respectively. In addition, later Ser expression in the presumptive vein cells is controlled by the Egfr pathway. Thus, a cis-regulatory element is sequentially regulated by multiple signaling pathways and a selector protein during Drosophila wing development. Such a mechanism is possibly conserved in the appendage outgrowth of other arthropods and vertebrates (Yan, 2004).

The results reported here demonstrate that a 794 bp cis-acting regulatory module in the Ser locus can be temporally regulated by three distinct mechanisms that are employed for the proper establishment of the DV organizer during wing development. (1) The selector protein Ap directly activates Ser expression in the dorsal compartment during the early third instar, which sets up N activation for the next stage. (2) By the middle of the third instar, the N pathway maintains Ser expression by a positive-feedback loop along the DV boundary. This feedback loop maintains Ser and Dl expression, leading to the activation of N signaling at the DV boundary, which is essential for establishing the DV organizer. (3) At the end of the third instar, as a result of Wg signaling, Ser is expressed in two stripes flanking the DV boundary, which limits N activation to the DV border. In addition, Ser expression in provein cells is dependent on input from the Egfr pathway. These results indicate how tissue-specific selector and signaling molecules can work sequentially to achieve a complex developmental process, such as organogenesis, which involves a complex temporal and spatial regulation of genes. However, the conclusion that the Ser minimal wing enhancer is sequentially regulated by Ap, Notch, Wg and Egfr does not exclude the possibility that these molecules/signaling pathways may cooperate and synergistically stimulate gene expression at certain stages. In this case, mutations that specifically impair response to the intended factor would affect Ser-lacZ expression in other phases of disc development (Yan, 2004).

In vitro and in vivo results suggest that the regulation of Ser by Wg signaling occurs directly through dTCF. Using DNase I footprinting, two major classes of dTCF binding sequences were found: the dTCF consensus sequence CCTTTGATCTT and the HMG consensus sequence WTTGWW, which are consistent with previously identified dTCF binding sequences. Interestingly, the presence of dTCF/HMG binding sites in the Ser minimal wing enhancer may explain the crosstalk observed between the 3' Ser enhancer and the 5' Ser promoter. HMG proteins can bend DNA, and could therefore bring the 3' enhancer close enough to interact with the transcriptional machinery binding at the 5' promoter (Yan, 2004).

In late third instar, Wg signaling is maintained in the DV organizer by the N pathway. Wg signaling activates Ser and Dl expression in the cells flanking the DV boundary, which in turn activates N signaling to maintain a positive-feedback loop between N and Wg signals. Because of an autonomous repression effect of N ligands on their receptor, Ser and Dl expression in the flanking cells also prevents N signaling from spreading out of the DV border. N signaling then turns off Ser and Dl expression by inducing cut at the border. Although the molecular nature of the dominant-negative effects of N ligands, and the repression of Ser and Dl by N signaling remains unknown, these mechanisms may play important roles in keeping the boundary sharp. Interestingly, the Ser minimal wing enhancer is also repressed at the DV border, suggesting that it is possible to study the molecular mechanism of Ser repression at the border using this 794 bp enhancer (Yan, 2004).

Given that the Ser-Fng-N pathway is evolutionarily conserved in appendage development between insects and vertebrates, the mechanism by which Ser is sequentially regulated by Ap, N, Wg and Egfr may also be conserved in appendage outgrowth of other arthropods and vertebrates. Consistent with this hypothesis, the Ap, Wg/Wnt and Egfr/Fgf pathways are also involved in appendage development in vertebrates, as well as D. melanogaster. Indeed, a BLAST search of the Drosophila pseudoobscura genome identified a putative homolog of the Ser minimal wing enhancer. Interestingly, this enhancer region is also located less than 1 kb downstream of the putative D. pseudoobscura Ser 3'UTR. Sequence comparisons between the Ser minimal wing enhancer from D. melanogaster and the putative D. pseudoobscura enhancer show a significant degree of similarity, whereas the similarities in the 5' and 3' flanking regions are lower. Importantly, sequences of putative Ap, Su(H) and dTCF binding sites are highly conserved in D. pseudoobscura and D. melanogaster. Although the strong conservation of sequence and location suggests that the putative D. pseudoobscura Ser enhancer may be a functional homolog of the D. melanogaster Ser minimal wing enhancer, it remains to be tested whether this enhancer drives reporter gene expression at the identical time and location in the D. melanogaster wing discs (Yan, 2004).

Border of Notch activity establishes a boundary between the two dorsal appendage tube cell types; Pangolin, a component of the Wingless pathway, is required for Broad expression and for rhomboid repression

Boundaries establish and maintain separate populations of cells critical for organ formation. Notch signaling establishes the boundary between two types of post-mitotic epithelial cells, the Rhomboid- and the Broad-positive cells. These cells will undergo morphogenetic movements to generate the two sides of a simple organ, the dorsal appendage tube of the Drosophila egg chamber. The boundary forms due to a difference in Notch levels in adjacent cells. The Notch expression pattern mimics the boundary; Notch levels are high in Rhomboid cells and low in Broad cells. Notch mutant clones generate an ectopic boundary: ectopic Rhomboid cells arise in Notch+ cells adjacent to the Notch mutant cells but not further away from the clonal border. Pangolin, a component of the Wingless pathway, is required for Broad expression and for rhomboid repression. It is further shown that Broad represses rhomboid cell autonomously. These data provide a foundation for understanding how a single row of Rhomboid cells arises adjacent to the Broad cells in the dorsal appendage primordia. Generating a boundary by the Notch pathway might constitute an evolutionarily conserved first step during organ formation in many tissues (Ward, 2006).

At the boundary, cells with high Notch express rhomboid, whereas cells with lower Notch express Broad. A new boundary is established at Notch mutant clone borders, where Notch+ cells adjacent to Notch cells ectopically express rhomboid and do not express Broad. Thus, in the dorsal anterior, when two cells with different Notch levels are adjacent to one another, the cell with higher Notch levels simultaneously represses Broad and promotes rhomboid expression. broad cells ectopically express rhomboid, indicating that Broad normally represses rhomboid expression. It is inferred that cells with higher Notch levels repress Broad, thereby allowing rhomboid expression. It is now proposed that when cells with different levels of Notch are located next to each other, the cells with high Notch repress Broad, allowing rhomboid expression. In contrast, cells with low Notch express Broad and therefore repress rhomboid expression (Ward, 2006).

Notch, an important modulator of boundary function in other tissues, establishes the boundary that defines the Rhomboid and the Broad dorsal appendage cell types. When Notch is removed from cells that should span the boundary, rhomboid is not expressed, and Broad is ectopically expressed. Thus, at the boundary, Notch regulates the patterning of both Rhomboid and Broad cell types. When Notch activity is removed from Region 1, ectopic Rhomboid cells (Notch+) arise adjacent to Notch (Broad) cells, thus resembling the normal Notch border. It is proposed that these Notch mutant clones produce ectopic borders of differential Notch activity, which in turn generate ectopic boundaries between Rhomboid and Broad domains (Ward, 2006).

Normally, Rhomboid cells arise all along the high–low Notch boundary in each dorsal appendage primordium. Based upon this observation, one might expect that Rhomboid cells would surround the Notch clones. In the current studies, however, it was found that only those cells close to the normal boundary turned on ectopic rhomboid. Two factors probably contribute to this result. First, other signaling pathways, most notably EGFR and DPP, are involved in specifying and positioning the Rhomboid and Broad cell populations within the follicular epithelium. Presumably, these other signaling pathways influence Broad/rhomboid expression in cells adjacent to Notch clones. Second, the ectopic Notch borders generated by Notch clones arise within the Broad domain, which normally has low levels of Notch. Therefore, many cells at the ectopic border may not have sufficient Notch activity to repress Broad and activate rhomboid (Ward, 2006).

Within the domain that would normally express Broad, loss of Notch causes the loss of Broad non-cell autonomously in adjacent cells and the appearance of ectopic rhomboid in these same cells. Furthermore, Notch clones spanning the boundary ectopically express Broad and do not express rhomboid. These findings are consistent with previous results demonstrating that dorsal appendage cells express either rhomboid or Broad, but never both markers. This work shows that broad cells ectopically express rhomboid, suggesting that one function of Broad in the follicular epithelium is to directly or indirectly repress rhomboid expression. Such regulation must occur (at least in part) in the 2.2-kb fragment that drives lacZ expression in a reporter construct. CONSITE software detects twenty Broad binding sites clustered together in this region; all four zinc-finger isoforms have the potential to bind. Thus, high levels of Broad could directly regulate rhomboid in Region 1. Additional work is needed to test this hypothesis (Ward, 2006).

Other factors must also regulate rhomboid expression in Region 2. Within clones spanning the boundary, ectopic expression of Broad prevents rhomboid expression. In cells adjacent to Notch clones, loss of Broad expression allows ectopic rhomboid expression. Nevertheless, the simple absence of Broad is insufficient to induce rhomboid expression, since the majority of cells in Region 2 lack Broad expression and do not express rhomboid. Presumably, high levels of EGFR and DPP signaling prevent rhomboid expression in these cells (Ward, 2006).

The Notch loss- and gain-of-function data, as well as the Notch expression pattern, all suggest that juxtaposition of two cells with different Notch levels is critical for establishing the boundary between Rhomboid and Broad cell types. How, then, is Notch protein level regulated? The restricted pattern of Notch in the dorsal anterior follicle cells suggests that Notch expression is determined by a combination of patterning instructions from DPP along the anterior/posterior axis and EGFR signaling along the dorsal/ventral axis (Ward, 2006).

The importance of regulating Notch protein levels is underscored by data showing that overexpression of full-length Notch represses Broad expression throughout the follicular epithelium. Since the full-length Notch receptor must be bound by ligand to initiate Notch signaling, a Notch ligand is either present throughout the follicular epithelium or is presented to the follicle cells by the underlying germ line. The Drosophila genome encodes two known Notch ligands, Delta and Serrate, and several potential ligands, such as CG9138. The absence of both Delta and Serrate in the follicular layer did not affect Broad or rhomboid expression. The function of other potential ligands in follicle cells is not currently known. It is also possible that the ligand for this process is present in the germ line. Delta is expressed in the germ line at the appropriate time and functions in the germ line to regulate follicle cell processes, such as the pinching-off of egg chambers in the germarium and the mitotic-to-endocycle transition at stage 7. Additionally, previous work demonstrates that egghead and brainiac, which encode modulators of Notch function, act in the germ line to pattern the dorsal anterior follicle cells. Regardless of the tissue distribution of the ligand, however, the ability to uniformly activate the Notch pathway throughout the follicle cell layer is note-worthy. This observation suggests that Notch levels, rather than spatial location of a ligand (or ligand modulator), determines where or how Notch signals in follicle cells of late stage egg chambers (Ward, 2006).

One of the most surprising aspects of the work presented here is that Notch clones act in a non-cell-autonomous manner to regulate Broad and rhomboid expression in adjacent cells. While surprising, non-cell-autonomous Notch activity occurs in the embryo, and most notably, at the D/V boundary in the wing disc. In the third-instar wing disc, Wingless is expressed in a 3- to 6-cell wide stripe spanning the D/V boundary, which separates the dorsal and ventral portions of the future wing blade. In this system, wingless-lacZ is repressed both within and adjacent to Notch clones. Thus, Notch clones act non-cell autonomously in two different tissues where boundaries act to distinguish different cell types (Ward, 2006).

What is the nature of the non-autonomous signal from the Notch clones? It is proposed two potential mechanisms to explain this process. First, Notch itself measures Notch levels in adjacent cells, either directly through homophilic adhesion or indirectly through interaction with Notch-binding proteins. When a Notch clone occurs in the dorsal anterior, adjacent cells sense the absence of Notch and respond as wild-type cells do when high-Notch cells neighbor low-Notch cells; they either repress Broad directly, or they repress Broad indirectly by affecting Pangolin (or some other component of the Wingless signaling pathway). Pangolin is needed to express Broad and therefore down-regulate rhomboid throughout the follicle cell layer. A second possibility is that when cells have little or no Notch activity, they might secrete an inhibitor of the Pangolin pathway that only affects cells with high Notch. The first mechanism is favored for its simplicity in accounting for rhomboid expression only at the border between high- and low-Notch-expressing cells (Ward, 2006).

The establishment of a border between Rhomboid and Broad cells is important for preventing intermingling of these cell types during tube formation (Ward, 2005). It is not clear, however, what mechanism separates the Broad and Rhomboid cells from each other at the border. In some situations, the non-transcriptional branch of the Notch pathway regulates F-actin (Major, 2005), which creates a “fence” that could help separate the two cell types from each other in the border. In dorsal anterior follicle cells, however, the canonical Notch pathway acts through the transcription factor Su(H). It is possible that in this cell type, the Notch pathway transcriptionally regulates a cell adhesion molecule or other component of an actin-binding protein complex, which in turn coordinates the cytoskeleton, thereby maintaining a separation between the Rhomboid cells and the Broad cells. Unlike cells at other boundaries in which an actin fence is evident, the Rhomboid and Broad cells undergo dramatic morphological changes and reorganize their actin networks to produce these effects. A fence that could maintain the separation of these cells during apical constriction, directed elongation, and convergent extension would be critical during these processes. One such Notch-interacting candidate gene that links to actin filaments is Echinoid. Future experiments will define whether Echinoid plays a role during border formation between Rhomboid and Broad cells (Ward, 2006).

Animals have a wide variety of organs containing different cell types arranged in a stereotypical manner. While the general morphogenesis of most organs has been described, little is known about the molecular mechanisms required to specify boundaries between diverse cell types and direct their subsequent reorganization to produce a functional structure. This study has shown that canonical Notch signaling is necessary to establish a boundary between the Broad and Rhomboid cells, which will form the dorsal and ventral portions of the dorsal appendage tube. Notch is also required in the vertebrate hindbrain for rhombomere boundary formation. Thus, in simple and more complex organs, Notch specifies boundaries between distinct cell populations needed for organ formation. Generating a boundary through Notch signaling could be an evolutionarily conserved first step during organ formation in many tissues. The next challenge is to define the molecular nature of the physical power that keeps the two different cell types separated from each other in the border (Ward, 2006).

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/Pangolin 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).

Molecular integration of Wingless, Decapentaplegic, and autoregulatory inputs into Distalless during Drosophila leg development

The development of the Drosophila leg requires both Decapentaplegic (Dpp) and Wingless (Wg), two signals that establish the proximo-distal (PD) axis by activating target genes such as Distalless (Dll). Dll expression in the leg depends on a Dpp- and Wg-dependent phase and a maintenance phase that is independent of these signals. This study shows that accurate Dll expression in the leg results from the synergistic interaction between two cis-regulatory elements. The Leg Trigger (LT) element directly integrates Wg and Dpp inputs and is active only in cells receiving high levels of both signals. The Maintenance (M) element is able to maintain Wg- and Dpp-independent expression, but only when in cis to LT. M, which includes the native Dll promoter, functions as an autoregulatory element by directly binding Dll. The 'trigger-maintenance' model describes a mechanism by which secreted morphogens act combinatorially to induce the stable expression of target genes (Estella, 2008).

This study provides evidence that Dll expression during Drosophila leg development is controlled by separate, synergistically interacting cis-regulatory elements. The first element, LT, activates transcription only in response to high levels of Wg and Dpp signaling. The second element, M, includes the Dll promoter and has the ability to activate transcription in a Wg- and Dpp-independent manner, but only when in cis to LT. Together, these results fit well with previous genetic experiments showing that the Wg and Dpp inputs into Dll are only required transiently, prior to ~60 hr AEL. Based on the data, it is hypothesized that LT, and perhaps other elements with similar properties, is responsible for activating the Wg- and Dpp-dependent phase of Dll expression. Further, the data suggest that the combination of LT+M executes the Wg- and Dpp-independent phase of Dll expression. The existence of a two-component cis-regulatory system for Dll expression has several interesting implications and provides a mechanistic understanding of how Wg, Dpp, and Dll inputs are integrated into Dll expression (Estella, 2008).

The requirement for multiple inputs for gene activation is a common theme in transcriptional regulation. Enhancer elements can be thought of as 'logic integrators' that are only active in the presence of the correct activators and in the absence of repressors. The LT element defined here behaves as such a logic integrator. To be active, at least three conditions must be met. (1) LT must be bound to a transcriptionally active form of Tcf, a condition which indicates high levels of Wg signaling. (2) LT must be bound to a transcriptionally active form of Mad, and, (3) LT must not be bound to Brk. The second and third of these three conditions both indicate high levels of Dpp signaling. This combination of inputs ensures that LT is triggered only only where Wg and Dpp signaling are both active. In addition, it is hypothesized that there must be another input that restricts LT's activity to the ventral discs (e.g., it is not active in other tissues where Wg and Dpp signaling intersect such as the wing disc). Such a ventral-specific input could be Dll itself, which is expressed before LT is active via the Dll304 enhancer, and/or another ventral-specific factor such as buttonhead (btd), which is also required for Dll expression. Consistent with this idea, LT-lacZ is lost in Dll clones and in Dll hypomorphic discs, suggesting that Dll input, in addition to Wg and Dpp, is required for its activity (data not shown) (Estella, 2008).

As noted above, Dpp signaling uses two mechanisms (Mad binding and absence of Brk) to control LT's activity. Because Brk, a transcriptional repressor, binds directly to LT, it restricts the domain in which Wg signaling can activate this element. This conclusion is best supported by the expression pattern of the LT reporter gene in which the Brk-binding sites were mutated. Specifically, the expression of this reporter (LTBrk–-lacZ) was expanded ventrally, indicating its potential to be activated more broadly by Wg signaling in the absence of this repressor. Thus, it is suggested that the primary role of Brk is to provide spatial information to LT activation. The absence of Brk, however, is apparently not sufficient for LT activation; Mad input into LT appears also to be essential. Several experiments support this conclusion. Most informatively, LT-lacZ was not expressed in Mad; brk clones, and LT-lacZ reporter genes with either Mad site mutated were not expressed in brk clones. Thus, even in the absence of Brk, LT requires Mad input. It is suggested that in contrast to providing spatial information, the Mad input into LT is important for boosting the level of its activation, together with Tcf, by providing an additional potent transcriptional activator. Further, LT is unlikely to be the only Dll cis-regulatory element that integrates Wg plus Dpp signaling during leg development. Although LT was the only fragment within the 14 kb of 5' DNA that drove strong expression in the leg disc in a standard reporter gene assay, thus allowing the dissection of Wg and Dpp signal integration, a second fragment was identified that was able to synergize with M to produce a Dll-like expression pattern. In summary, these data suggest that during the Wg- and Dpp-dependent stage, Dll expression is regulated by the direct binding of Tcf, Mad, and Brk to LT and, perhaps, additional regulatory elements (Estella, 2008).

As is the case for Dll, there are examples of other genes that have separable initiation and maintenance phases of expression. For many of these examples, expression is maintained by the trxG and PcG of epigenetic regulators. There are also examples of genes that require enhancer-promoter communication for maintenance. For example, a regulatory element from the Hoxb4 gene requires sequences from its own promoter for stable expression in the mouse hindbrain. In this case, a key input into the promoter-proximal sequences is the PcG protein, YY1. Dll expression is unaffected in trx mutant clones but is lost in a subset of Pc and Scm clones, raising the possibility that PcG functions play a role in maintenance. However, PcG functions are more typically associated with maintaining genes in a repressed state, not an expressed state. Moreover, because of PcG's widespread role in gene silencing, many genes are likely to be derepressed in these clones. In fact, the Hox gene Abd-B is derepressed in these clones, and Abd-B has the ability to repress Dll. Thus, on balance, it seems more likely that the loss of Dll expression observed in some Pc clones is an indirect effect. In contrast, the results strongly argue that positive autoregulation, by direct binding of Dll to the M element, plays an important role in Dll maintenance (Estella, 2008).

One conclusion drawn from these observation that both LT and M are required for maintenance is that LT requires the Dll promoter to be fully active. Such promoter-specific enhancer activation has been observed previously and is generally thought to be important for remote enhancers to stimulate transcription from the correct promoter in gene-dense regions of the genome. The LT+M synergy described in this study is distinct from these other examples. In this case, although enhancer-promoter compatibility may be part of the reason that LT works better with M (and over large distances), the results show that the combination of the two has properties that are not exhibited by either element on its own. Specifically, while M-lacZ is very weakly expressed in leg discs, and LT-lacZ requires continuous Wg and Dpp inputs, the combination of LT+M allows Dll autoregulation to occur in a Wg- and Dpp-independent manner. Moreover, LT+M is not simply a Dll autoregulatory element: even though Dll is expressed in the wing disc, transcriptional activation by LT+M remains restricted to the ventral imaginal discs. This observation implies that the Dll input into LT+M can only occur in cells where LT was activated, which itself only happens in ventral discs. Thus, LT+M is not only a two-component Dll autoregulatory element, but is an autoregulatory element that requires the prior Wg- and Dpp activation of LT (Estella, 2008).

These observations lead to the suggestion of two classes of models by which maintenance may occur. In one, an activated LT element changes the chromatin structure of M, for example, by changing the position of a repressive nucleosome so that it can function as an autoregulatory element. According to this model, the continued presence of LT is required to maintain this chromatin structure. A second model that would also accommodate these data is that the combination of LT plus M is required to increase the efficacy of transcriptional activation by, for example, providing additional Dll (or other activator) binding. According to this scenario, LT activation by Wg and Dpp triggers the initial interaction between the LT and M elements, which would then be stabilized in a Wg- and Dpp-independent manner. These models are not mutually exclusive and both can be tested by analyzing the chromatin status at the M and LT elements (Estella, 2008).

The results also raise the question of what purpose this two-step trigger-maintenance mechanism may serve. One possibility is that, by having only a transient requirement for Wg and Dpp, these morphogens are available for carrying out completely different tasks, without affecting Dll expression. In support of this idea, in addition to working together to create the PD axis, Wg and Dpp function independently to instruct ventral and dorsal leg fates, respectively. Some of these late Wg and Dpp patterning functions may also require Dll input. The trigger-maintenance logic described in this study in principle allows Wg and Dpp to execute functions in collaboration with their own downstream target, Dll (Estella, 2008).

It is also noteworthy that the transient nature of the Wg and Dpp inputs into Dll is not the typical way these morphogens regulate their target genes in other tissues. In the Drosophila wing, for example, Dpp and Wg are required to continuously activate their targets, such as vestigial, optormotor blind, and spalt. One signficant difference between the regulation of wing and leg target genes by these morphogens is that in the wing Wg and Dpp generally act independently, whereas in the leg they act combinatorially to activate PD genes. Specifically, although they are expressed in ventral and dorsal sectors, respectively, Wg and Dpp activate Dll and dac in circular or nearly circular domains whose centers are located where the Dpp and Wg expression domains touch, in the middle of the leg disc. The trigger-maintenance mechanism defined in this study avoids the need for target genes such as Dll to continuously integrate Wg and Dpp inputs as the disc grows in size, and provides a mechanism to generate circular domains of gene expression using dorsal and ventral morphogen inputs (Estella, 2008).

Wingless signaling directly regulates cyclin E expression in proliferating embryonic PNS precursor cells

Cell proliferation and cell type specification are coordinately regulated during normal development. Cyclin E, a key G1/S cell cycle regulator, is regulated by multiple tissue-specific enhancers resulting in dynamic expression during Drosophila development. This study further characterized the enhancer that regulates cyclin E expression in the developing peripheral nervous system (PNS) and shows that multiple sequence elements are required for the full cyclin E PNS enhancer activity. Wg signaling is important for the expression of cyclin E in the sensory organ precursor (SOP) cells through two conserved TCF binding sites. Blocking Wg signaling does not completely block SOP cell formation but does completely block SOP cell proliferation as well as the subsequent differentiation (Deb, 2008).

The results reveal that cyclin E expression in developing PNS precursor cells is regulated by a large enhancer containing multiple sequence elements, including two TCF-binding sites that mediate the regulation by Wg signaling. While these TCF-binding elements are essential for the activity of the PNS enhancer, proximal and distal elements in the 4.6-PNS sequence appear to be important for full activity. The importance of Wg in the regulation of the PNS expression of cyclin E is supported by the fact that wg mutant embryos displayed decreased cyclin E expression in the developing PNS cells. This reduction in cyclin E expression in wg mutant embryos was accompanied by an inhibition of BrdU incorporation in the developing PNS, and an inhibition of the determination of the Pros and Elav expression cells in the developing PNS. It is possible that the block in differentiation into the Pros and Elav positive cells is a consequence of the inhibition of cyclin E expression or perturbations to the cell proliferation. However it is also possible that the observed differentiation block in PNS is due to a function of Wg that is independent of PNS cell proliferation. Further studies will be needed to resolve this issue (Deb, 2008).

In addition to wg, a number of other mutations such as achaete/scute (ac/sc) complex and da have also been reported to block PNS precursor proliferation and affect the expression of several cell cycle genes. Ac/Sc complex proteins and Da are bHLH proteins that are important in all aspects of es-PNS precursor differentiation while bHLH protein Atonal (ato) and Da are required for all aspects of ch-PNS precursor development. Recent studies of the expression of the Cdk inhibitor Dap during cell type specification revealed that Dap expression is directly regulated by the same developmental mechanisms that control the differentiation of these cell types. Therefore it will be interesting to test if bHLH proteins such as Da also directly regulate cyclin E expression in the developing PNS cells (Deb, 2008).

Bipartite recognition of DNA by TCF/Pangolin is remarkably flexible and contributes to transcriptional responsiveness and tissue specificity of Wingless signaling

The T-cell factor (TCF) family of transcription factors are major mediators of Wnt/beta-catenin signaling in metazoans. All TCFs contain a High Mobility Group (HMG) domain that possesses specific DNA binding activity. In addition, many TCFs contain a second DNA binding domain, the C-clamp, which binds to DNA motifs referred to as Helper sites. While HMG and Helper sites are both important for the activation of several Wnt dependent cis-regulatory modules (W-CRMs), the rules of what constitutes a functional HMG-Helper site pair are unknown. This study employed a combination of in vitro binding, reporter gene analysis and bioinformatics to address this question, using the Drosophila family member TCF/Pangolin (TCF/Pan) as a model. It was found that while there are constraints for the orientation and spacing of HMG-Helper pairs, the presence of a Helper site near a HMG site in any orientation increases binding and transcriptional response, with some orientations displaying tissue-specific patterns. Altering an HMG-Helper site pair from a sub-optimal to optimal orientation/spacing dramatically increases the responsiveness of a W-CRM in several fly tissues. In addition, the knowledge gained was used to bioinformatically identify two novel W-CRMs, one that was activated by Wnt/beta-catenin signaling in the prothoracic gland, a tissue not previously connected to this pathway. In sum, this work extends the importance of Helper sites in fly W-CRMs and suggests that the type of HMG-Helper pair is a major factor in setting the threshold for Wnt activation and tissue-responsiveness (Archbold, 2014 PubMed).

Wnt-mediated repression via bipartite DNA recognition by TCF in the Drosophila hematopoietic system

The Wnt/beta-catenin signaling pathway plays many important roles in animal development, tissue homeostasis and human disease. Transcription factors of the TCF family mediate many Wnt transcriptional responses, promoting signal-dependent activation or repression of target gene expression. The mechanism of this specificity is poorly understood. Previous work has demonstrated that for activated targets in Drosophila, TCF/Pangolin (the fly TCF) recognizes regulatory DNA through two DNA binding domains, with the High Mobility Group (HMG) domain binding HMG sites and the adjacent C-clamp domain binding Helper sites. This study reports that TCF/Pangolin utilizes a similar bipartite mechanism to recognize and regulate several Wnt-repressed targets, but through HMG and Helper sites whose sequences are distinct from those found in activated targets. The type of HMG and Helper sites is sufficient to direct activation or repression of Wnt regulated cis-regulatory modules, and protease digestion studies suggest that TCF/Pangolin adopts distinct conformations when bound to either HMG-Helper site pair. This repressive mechanism occurs in the fly lymph gland, the larval hematopoietic organ, where Wnt/beta-catenin signaling controls prohemocytic differentiation. This study provides a paradigm for direct repression of target gene expression by Wnt/beta-catenin signaling and allosteric regulation of a transcription factor by DNA (Zhang, 2014; PubMed).

pangolin: Biological Overview | Evolutionary Homologs | Protein Interactions | Developmental Biology | Effects of Mutation | References

Home page: The Interactive Fly © 1997 Thomas B. Brody, Ph.D.

The Interactive Fly resides on the
Society for Developmental Biology's Web server.