cubitus interruptus


Targets of Activity

The identity of anterior cells in the wing imaginal disc requires ci function. Anterior cells lacking ci express hedgehog and adopt posterior properties without expressing engrailed. Most clones cause an up-regulation of CI protein levels in surrounding cells, in a manner that is similar to that of CI along the A/P compartment boundary. Increased levels of CI can induce the expression of the HH target gene decapentaplegic in a HH-independent manner, suggesting that dpp is a target gene of CI. This is the first identified component of the HH-signaling cascade that is able to activate dpp transcription. Also, there is reason to believe that CI can also repress dpp, suggesting that CI can act as both a repressor and as an activator of dpp transcription in a concentration dependent manner. CI also positively regulates patched. Thus, expression of CI in anterior cells controls limb development by restriction HH transcription to posterior cells and by conferring competence to respond to HH by mediating the transduction of this signal. The multiple role of CI in the anterior compartment suggests that anterior cell identity is not a default fate that imaginal cells adopt in the absence of engrailed (Domínguez, 1996).

Engrailed and Cubitus interruptus regulate patched. Early ubiquitous expression of patched is followed by its repression in the posterior portion of each parasegment; subsequently each broad band of expression splits into two narrow stripes. The first step in patched regulation is under the control of Engrailed whereas the second requires the activity of both cubitus interruptus and patched itself. Furthermore, the products of engrailed, wingless and hedgehog are essential for maintaining the normal pattern of expression of patched (Hidalgo, 1990).

Epistasis analysis indicates that cubitus interruptus functions in the Hedgehog (HH) signal transduction pathway and is required to maintain wingless expression in the embryo. Ectopic expression of ci in imaginal discs and the embryo activates the expression of HH target genes. One of these target genes, patched, forms a negative feedback loop with ci that is regulated by HH signal transduction. Activation is also achieved using the CI zinc finger domain fused to a heterologous transactivation domain. Conversely, repression of HH target genes occurs in animals expressing the CI zinc finger domain fused to a repression domain. Regions of the CI protein that are responsible for its ability to transactivate and its subcellular distribution have been identified. Sequences C terminal to the zinc finger domain are required for transactivation and to regulate the subcellular distribution of CI protein; a deleted C terminal region is distributed uniformly throughout the nucleus and the cytoplasm, while wild-type CI is primarily cytoplasmic (Hepker, 1997)

Cubitus interruptus is required to maintain expression of the wingless gene and to specify naked cuticle within each epidermal segment (Motzny, 1995).

Misexpression of CI in the Engrailed domain (by placing CI under the control of an en promoter) activates wingless transcription. The expression domains of wingless in such embryos are significantly broader than in wild-type cells. Placing ci under the control of the hairy promoter (hairy is expressed only in alternating parasegments), and testing such a construct in hh mutant embryos, results in a pair-rule phenotype, where every other segment shows naked cuticle. In these embryos, wg expression is present in alternate parasegments. This provides conclusive evidence the CI regulates wingless (Alexandre, 1996).

A wingless enhancer region has been described whose Cubitus interruptus (Ci) binding sites mediate Ci-dependent transcriptional activation in transiently transfected cells. Hedgehog (Hh) and Patched (Ptc) act through those Ci binding sites to modulate the level of Ci-dependent transcriptional activation in S2 cells. To test for effects of Ptc and Hh, titrations of Ci cDNA in cultured cells were performed on an expression vector regulated by this enhancer region. The titrations were performed either in the presence of Hh cDNA or in the presence of Ptc cDNA. Reporter activity is reduced 3-fold in the presence of co-transfected Ptc. The addition of Hh results in a 1.5-fold increase in reporter activity over that observed for Ci alone. This same wg enhancer region is Hh responsive in vivo and its Ci binding sites are necessary for its activity. This provides strong evidence that Hh affects wg transcription through post-translational activation of Ci (Von Ohlen, 1997a).

Patterning of the Drosophila embryo depends on the accurate expression of wingless (wg), which encodes a secreted signal required for segmentation and many other processes. Early expression of wg is regulated by the nuclear proteins of the gap and pair-rule gene classes but, after gastrulation, wg transcription is also dependent on cell-cell communication. Signaling to the Wg-producing cells is mediated by the secreted protein, Hedgehog (Hh), and by Cubitus interruptus (Ci), a transcriptional effector of the Hh signal transduction pathway. The transmembrane protein Patched (Ptc) acts as a negative regulator of wg expression; ptc- embryos exhibit ectopic wg expression. According to the current models, Ptc is a receptor for Hh. The default activity of Ptc is to inhibit Ci function; when Ptc binds Hh, this inhibition is released and Ci can control wg transcription. An investigation was carried out of the cis-acting sequences that regulate wg during the time that wg expression depends on Hh signaling. A region consisting of 4.5 kb immediately upstream of the wg transcription unit can direct expression of the reporter gene lacZ in domains similar to the normal wg pattern in the embryonic ectoderm. Expression of this reporter construct expands in ptc mutants and responds to hh activity. Within this 4.5 kb, a 150 bp element, highly conserved between D. melanogaster and Drosophila virilis, is required to spatially restrict wg transcription. Activity of this element depends on ptc, but it contains no consensus Ci-binding sites. The 150 bp G box, 91% identical with its counterart from D. virilis, mediates repression of wg in a ptc-dependent manner. The G box is sufficient for conferring wild-type width to reporter stripes, which in turn expand in a ptc mutant background, thus behaving like wg itself. Deletion of element G result in wide stripes in a wild-type embryo, suggesting that this is a binding site for a transcriptional repressor active in cells anterior to the wild-type wg domain. A repressor that binds element G could possibly act in parallel to ptc and hh; in such a case, the repressor's activity would be overcome by an Hh-regulated activator, i.e. Ci. The simpler explanation is that a repressor functions as another endpoint of Hh signaling. The discovery of an element that is likely to bind a transcriptional repressor was unexpected, since the prevailing model suggests that wg expression is principally controlled by Hh signaling acting through the Ci activator. It is shown that wg regulatory DNA can drive lacZ in a proper wg-like pattern without any conserved Ci-binding sites (Lessing, 1998).

Direct wg autoregulation (autocrine signalling) is masked by its paracrine role in maintaining hh, which in turn maintains wg. shaggy/zeste-white3 and patched mutant backgrounds have been used to genetically uncouple this positive-feedback loop and to study autocrine wg signalling. Direct wg autoregulation differs from wg signalling to adjacent cells in the importance of fused, smoothened and ci relative to zw3 and armadillo. wg autoregulation during this early hh-dependent phase differs from later wg autoregulation by lack of gooseberry participation (Hooper, 1994).

The effects of ectopic CI on decapentaplegic and patched transcription was assayed using dpp and ptc reporter plasmids. In the third larval instar wing disc, expression of the dpp reporter is activated ectopically in all cells expressing high levels of CI protein in the anterior compartment, but is not activated in the posterior compartment. Expression of the ptc reporter is activate ectopically throughout both anterior and posterior compartments. Thus in the embryo, high levels of CI protein are sufficient to activate transcription of patched, even on the presence of Engrailed; however, ectopic CI activity apparently cannot overcome the repression of dpp transcription by EN (Alexandre, 1996).

araucan and caupolican, two members of the iroquois gene complex, are highly related proteins belonging to a new family of homeoproteins. ARA and CAUP regulate the pattern elements (sensory organs and veins) in wing imaginal discs by spatially restricting domains of expression of the proneural genes achaete and scute and the provein gene rhomboid. ara-caup expression is restricted to two symmetrical patches located one at each side of the dorsoventral compartment border. ara-caup expression in these patches is necessary for the specification of the prospective vein L3 and associated sensory organs. Here, ara-caup expression is mediated by the Hedgehog signal through its induction of high levels of Cubitus interruptus in anterior cells near the the AP compartment border. The high levels of CI activate decapentaplegic expression, and together, CI and DPP positively control ara-caup. patched overexpression is equivalent to a reduced hh function in that accumulation of CI and DPP at the AP border are strongly depressed. The wing pouch of patched mutants have much reduced or absent ara-caup L3 patches. dpp by itself is insufficient to account for ara-caup expression. wingless is expressed in a narrow stripe of cells that stradles the DV compartment boundary of the wing disc corresponding to the prospective wing margin. The dorsal and ventral ara-caup L3 patches are separated by a gap that corresponds to the cells that detectably accumulate WG. Clones of mutant wg expressing cells spanning the gap between the L3 patches extend these patches toward the DV border and a narrow gap of only one or two cell diameters remains. Thus WG represses ara-caup expression at the prospective wing margin domain. Likewise repression by Engrailed is most likely to be responsible for the posterior border of ara-caup expression in the L3 patches (Gómez-Skarmeta, 1996).

The COE transcription factor Collier is a mediator of short-range Hedgehog-induced patterning of the Drosophila wing: Ci controls the formation of the central intervein by activating the expression of blistered

The secreted Hedgehog (Hh) proteins have been implicated as mediators of positional information in vertebrates and invertebrates. A gradient of Hh activity contributes to antero-posterior (A/P) patterning of the fly wing. In addition to inducing localised expression of Decapentaplegic (Dpp), which in turn relays patterning cues at long range, Hh directly patterns the central region of the wing. Short-range, dose-dependent Hh activity is mediated by activation of the transcription factor Collier (Col). In the absence of col activity, longitudinal veins 3 and 4 (L3 and L4) are apposed and the central intervein is missing. Hh expression induces col expression in a narrow stripe of cells along the A/P boundary through a dual-input mechanism: inhibition of proteolysis of Cubitus-interruptus (Ci) and activation of the Fused (Fu) kinase. Col, in cooperation with Ci, controls the formation of the central intervein by activating the expression of blistered (bs), which encodes the Drosophila serum response factor (D-SRF). D-SRF activity is required for the adoption and maintenance of the intervein cell fate. Furthermore, col is allelic to knot, a gene involved in the formation of the central part of the wing. This finding completes an understanding of the sectorial organization of the Drosophila wing. It is concluded that Col, the Drosophila member of the COE family (Col/Olf-1/EBF) of non-basic, helix-loop-helix (HLH)-containing transcription factors, is a mediator of the short-range organizing activity of Hh in the Drosophila wing. These results support the idea that Hh controls target gene expression in a concentration-dependent manner and highlight the importance of the Fu kinase in this differential regulation. The high degree of evolutionary conservation of the COE proteins and the diversity of developmental processes controlled by Hh signaling raises the possibility that the specific genetic interactions depicted here may also operate in vertebrates (Vervoort, 1999).

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) 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).

The expression of dppho-lacZ was examined throughout larval development. Expression of the dppho-lacZ reporter diminishes with time. Expression is detected as a contiguous stripe at second larval instar but fades in intensity by mid third instar. A likely explanation for this observation is that this reporter lacks the sequences required for maintained expression. dpp autoregulation has been reported in other tissues providing a plausible mechanism for maintenance of expression. Because binding sites for downstream transducers of dpp are present in the dppho enhancer, the response of dppho-lacZ to ectopic expression of dpp was examined. A 30AGAL4 driver was used to induce expression of a UAS-dpp transgene in a dppho-lacZ background. No animals showed any response by dppho-lacZ to ectopic induction of the Dpp signal. To determine whether sequences adjacent to the dppho enhancer element were required for dpp autoregulation, expression of the dppho-lacZ reporter line was compared to a reporter containing dppho plus an additional 2.5 kb of flanking sequences (BS3.2). The BS3.2 reporter directs expression of an A stripe along the A/P boundary that extends into the notum and is robust both early (second instar) and late (late third instar). These two reporters were assayed in a heterozygous dpp hypomorphic background. Expression of dppho-lacZ is unaffected while BS3.2 is expressed at wild-type levels early but fades by the end of third larval instar, suggesting that the larger reporter is sensitive to dpp autoregulation. Consistent with this idea, the expression levels of the BS3.2 reporter increase in response to ectopic UAS-dpp driven by 30AGAL4 but only at the A/P boundary where expression of 30AGAL4 overlaps with elevated full-length Ci (Hepker, 1999).

Hedgehog, acting through Ci, creates a gradient of DPP activity in Drosophila wing imaginal discs

Hedgehog (Hh) and Decapentaplegic (Dpp) direct anteroposterior patterning in the developing Drosophila wing by functioning as short- and long-range morphogens, respectively. The activity of Dpp is graded and is directly regulated by a novel Hh-dependent mechanism. Dpp activity is monitored by visualizing the activated form of Mothers against dpp (Mad), a cytoplasmic transducer of Dpp signaling. Activated Mad levels are highest near the source of Dpp but are unexpectedly low in the cells that express dpp. Hh induces dpp in these cells; it also attenuates their response to Dpp by downregulating expression of the Dpp receptor thick veins (tkv). It has been suggested that regulation of tkv by Hh is a key part of the mechanism that controls the level and distribution of Dpp (Tanimoto, 2000).

To determine whether the low levels of phosphorylated Mad (p-MAD) in dpp-expressing cells reflect an autoregulatory influence on Dpp signaling that functions only in dpp-expressing cells, or whether p-MAD levels are controlled by another signal such as Hh, p-MAD was monitored in wing discs carrying clones that ectopically express hh or dpp. If Dpp has the potential to attenuate its own signal transduction, the level of p-MAD would be expected to decrease cell autonomously in cells that express dpp. However, the level of p-MAD is elevated around the clones with no obvious reduction within the clones. This observation is not consistent with an autoregulatory mechanism. In contrast, clones expressing hh in the A compartment cause both a nonautonomous reduction of p-MAD level as well as ectopic elevation of p-MAD around the clones, which is due to ectopic induction of dpp by Hh. No changes in p-MAD levels are observed when Hh is expressed in the P compartment. These observations suggest that Hh attenuates Dpp signal transduction by regulating the type I receptor-dependent phosphorylation of MAD. Thus, the activity of Dpp depends on both the concentration of Dpp and Hh (Tanimoto, 2000).

It was next asked whether Hh directly controls p-MAD levels. First, clones that ectopically express HhCD2, a membrane-tethered form of Hh, were compared to clones that express a diffusible form of Hh. P-MAD levels are reduced both in cells that expressed HhCD2 and in cells that are immediately adjacent. In contrast, the levels of p-MAD are reduced more broadly when cells express diffusible Hh. The distribution of p-MAD was examined when clones of cells mutant for either Protein kinase A (Pka) alone or both Pka and dpp were induced. Pka functions to antagonize Hh signaling: Pka- clones in the A compartment fail to repress Hh signaling in a cell-autonomous manner. Pka- clones in the A compartment have reduced levels of p-MAD. Moreover, p-MAD accumulates to high levels in cells surrounding Pka- clones, due, presumably, to the ectopic induction of Dpp by constitutive Hh signaling in the mutant cells. In clones mutant for both Pka and dpp, the reduction was evident, but it was not accompanied by elevated levels in the surrounding cells. Outside the double-mutant clones, p-MAD appears to respect the endogenous gradient of Dpp. Autonomous reduction in the level of Spalt is also seen in both types of mutant clones, confirming both that p-MAD accurately manifests the active state of Dpp signaling and that the reduction of p-MAD leads to the decrease of target gene expression. It is concluded that Hh signaling directly attenuates Dpp signal transduction regardless of the level of endogenous Dpp (Tanimoto, 2000).

The contribution of Cubitus interruptus (Ci) to Hh-dependent attenuation of Dpp signaling was examined. ci is expressed in the A compartment only. In clones that ectopically express Ci in the P compartment where Hh is abundant, the levels of p-MAD were reduced in a cell-autonomous manner. Levels of Sal are similarly reduced in these cells. Clones that ectopically express ci in A compartment regions that receive little or no Hh have no effect on p-MAD levels. It is concluded that Hh both induces dpp expression and downregulates Dpp signal transduction in the same cells (Tanimoto, 2000).

Because Ci is involved in regulating p-MAD levels, it is possible that Hh controls the expression of a component that functions upstream of Mad phosphorylation. The possibiliy that Hh regulates the transcription of the Dpp receptors was therefore investigated. Dpp preferentially signals through the Tkv receptor and also negatively regulates tkv expression. Therefore, the correlation between tkv expression and the activity of Dpp was examined. The level of tkv expression is higher in cells located peripherally and is lower in the central region. In addition, a sharp reduction in expression at the A/P border was observed, a pattern very similar to that of p-MAD. The level of tkv expression in the area between the peripheral region and the A/P border is referred to as 'basal' and the reduced level at the A/P border as 'hyperrepression'. Interestingly, the basal level in the P compartment is higher than it is in the A compartment. This may account for the steeper gradient of p-MAD in the P compartment, since high levels of Tkv limit the movement of Dpp; since the spread of Dpp would be less in the P compartment, its gradient of activity would be expected to be steeper (Tanimoto, 2000).

In order to investigate the role of Dpp in generating the complex pattern of tkv expression, tkv and p-MAD were monitored in discs that ubiquitously express dpp. These discs exhibit significant overgrowth. tkv-lacZ at the A/P border remains hyperrepressed, but the level of peripheral tkv is significantly reduced, apparently to the basal level. This suggests that the level of tkv in the peripheral regions is directly regulated by Dpp but that the level at the A/P compartment border is not. The basal level in the P compartment remains higher than in the A compartment. Distribution of p-MAD is consistent with the expression pattern of tkv: overexpression of dpp caused the ubiquitous elevation of p-MAD, except at the A/P border. In contrast, phosphorylation of Mad at the A/P border is unchanged in the presence of excess Dpp. This suggests that the level of tkv along the A/P border is limiting. When constitutively active Tkv (tkv*) is ubiquitously expressed, peripheral tkv expression is reduced to the basal level, comparable to Dpp overexpression. Unlike the discs in which Dpp is overexpressed, no reduction in p-MAD is observed at the A/P border in these discs. These results suggest that in middle regions of the A and P compartments in normal discs, Dpp signaling reduces tkv expression from the levels at the periphery to the basal level. However, Dpp signaling is not responsible for the hyperrepression at the A/P border or for the difference between the basal levels in the A and P compartments. Given that hyperrepression of p-MAD along the A/P border is not observed in the presence of TKV*, it is more likely that in this region, Hh modulates p-MAD levels by regulating tkv (Tanimoto, 2000).

To ask whether Hh controls tkv expression directly at the A/P border, clones of cells mutant for patched (ptc), expressed in all A cells, were examined. The Hh signal transduction is constitutively active in the absence of the ptc activity. p-MAD and tkv-lacZ were monitored in ptc- clones. The clones in the A compartment ectopically activate Hh signaling in a cell-autonomous manner; they also caused cell-autonomous repression of tkv. Outside of the clone, the level of tkv is reduced to the basal level, but this level is higher than within the clone. This behavior is likely due to ectopic expression of dpp. P-MAD levels are also reduced in the clone and are higher around the clone. This establishes that Hh signaling directly represses tkv and, as a consequence, represses phosphorylation of MAD. This conclusion is confirmed by examining discs carrying clones of cells in the P compartment that ectopically express ci. tkv is autonomously repressed in these clones, indicating that Ci-mediated Hh signaling directly represses tkv (Tanimoto, 2000).

In order to understand the significance of Hh-dependent repression of tkv, the level of tkv expression was changed. In the wing disc, when overexpressing tkv in the dorsal compartment under the control of apterous (ap) enhancer, the levels of the dorsal p-MAD and Spalt expression at the A/P border are elevated, and their distribution is significantly narrower than in the ventral compartment. This is probably due to the sequestration of Dpp protein by the elevated Tkv. In addition, the adult wing misexpressing tkv was examined in dpp-expressing cells where tkv is hyperrepressed. The overexpression caused small wings with abnormal patterning in the central region of the wing. These results are consistent with the proposal that Hh, but not Dpp, patterns the central wing region. It should be noted, however, that the level of tkv expression in the experimental clones is probably at least several fold higher than normal, since these experiments utilized the Gal4/UAS system (Tanimoto, 2000).

The activities of Dpp were examined when tkv expression differed from wild type by only 2-fold. Clones mutant for tkv were generated in a heterozygous background such that both mutant (0 copies of the wild-type tkv) and fully wild-type sister clones (2 copies of wild-type tkv) were produced. However, since clones of cells with no Tkv activity do not survive in the wing pouch because they need Tkv activity to grow, only their sister clones survive. Homozygous (+/+) clones at the A/P border increase p-MAD and Spalt levels autonomously. The same analysis was performed using a null sax allele: differences in the levels of p-MAD and Spalt between clones carrying two wild-type sax and cells lacking one copy of sax were negligible. Taken together, it is proposed that the precise regulation of the Tkv receptor level by Hh signal is necessary for Dpp morphogen to shape the correct activity gradient (Tanimoto, 2000).

The repressor and activator forms of Cubitus interruptus control Hedgehog target genes through common generic Gli-binding sites

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

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

For the minimal dpp and brinker enhancers, a Hh-independent activator input has been postulated that causes a basal expression level. This transcriptional activity can either be synergistically enhanced by Ci[act] or suppressed by Ci[rep], depending on which form of Ci is prevailing and thus predominantly binding to the Gli/Ci-binding site. This scenario represents the simplest case of a Hh target gene, one that responds to both forms of Ci. To achieve selective responsiveness to only one form of Ci, as in the cases of hh and ptc, additional cis-acting elements must have evolved. At present, the cis elements that make some Ci target genes different from others are unknown. However, a firm case can be made for the relevance of this yet unknown mechanism. Let’s consider first the hh gene, which is particularly interesting because it responds effectively and selectively to Ci[rep]. hh expression levels can neither be increased in the posterior compartment nor ectopically induced in the anterior compartment by overexpression of constitutively active forms of Ci. But even very low levels of Ci[rep] suffice to repress hh expression in anterior cells near the AP boundary, despite the presence of high levels of Ci[act]. This was revealed by the observation that anterior ci mutant clones located close to the AP boundary ectopically express the hh gene. If Ci controls hh directly, by binding to Gli sites of the hh gene, the hh promoter must be configured to assemble a transcriptional complex that is unable to effectively interact with Ci[act] (Muller, 2000 and references therein).

The converse situation is found for ptc, which is regulated by Hh exclusively via Ci[act] and for which a direct binding of Ci to enhancer elements has been shown. The ptc gene is normally ‘off’ in P compartment cells but can readily be induced by expressing Ci ectopically. Similarly, the low expression levels found in A compartment cells can be augmented by ectopically providing Ci[act]. But this low level expression of ptc is not controlled by Ci[rep]. ci mutant clones in anterior regions, where Ci[rep] is the predominant form of Ci, show no increase in ptc expression. In addition, overexpression of Ci[rep] in A cells does not reduce the low levels of ptc. The arrangement of transcription factors on the ptc promoter must either facilitate the binding of Ci[act] versus Ci[rep], or they must be largely insensitive to Ci[rep] activity. If this were not the case and the ptc gene would be effectively repressed by Ci[rep], insufficient levels of Ptc protein would cause Hh-independent Smo signalling. This in turn would prevent the formation of Ci[rep] which plays a critical role in the repression of genes such as dpp and hh. Therefore an important question that remains to be answered in the future is how Ci targets, such as hh and ptc, evolved their selective responsiveness to Ci through distant cis-regulatory elements (Muller, 2000).

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

Groucho mediates a Ci-independent mechanism of hedgehog repression in the anterior wing pouch

A function of Gro in imaginal development has been investigated, namely the repression of hedgehog in anterior wing pouch cells. hh is repressed in anterior compartments at least partly via Ci[rep], a form of the multifunctional transcription factor Cubitus interruptus (Ci). Cells in the wing primordium close to the AP boundary need gro activity to maintain repression of hh transcription, whereas in more anterior cells gro is dispensable. This repressive function of Gro does not appear to be mediated by Ci[rep]. Analysis of mutant gro transgenes has revealed that the Q and WD40 domains are both necessary for hh repression. Yet, deletion of the WD40 repeats does not always abolish Gro activity. These findings provide new insights both into the mechanisms of AP patterning of the wing and into the function of Gro (Apidianakis, 2001).

Although Ci[rep]-mediated repression can account for the lack of hh expression away from the AP boundary, it has not been firmly established that Ci[rep] is operational close to the AP boundary. These cells receive high Hh signal and as a result not only do they not process Ci to Ci[rep], but also they activate full-length Ci into a strong activator, Ci[act], by post-translational modification. There is indirect evidence that Hh-receiving cells do not contain sufficient Ci[rep] levels to repress hh: in posterior cells, ci is repressed by En; other than this, the cellular mechanism for Hh signal transduction is present. When full-length ci is provided by ectopic expression in the posterior compartment, hh-lacZ is not repressed. This suggests that these cells cannot produce appreciable amounts of Ci[rep], consistent with their responding to Hh signaling. That this is indeed the case was shown by the fact that ectopic expression of ci does repress posterior hh-lacZ in smo loss-of-function clones, where the Hh signal transduction has been disrupted. If anterior cells that are exposed to Hh behave similarly, then the lack of hh expression there cannot be attributed to Ci[rep]. It is proposed that a Gro-dependent repression complex supplies this function, since gro- clones exhibit strong derepression of hh-lacZ near the AP boundary. The Gro complex is not required in anterior cells far from the boundary, because those receive no Hh signal and thus contain sufficient Ci[rep] to repress hh. Accordingly, by supplying increased levels of Ci[rep] near the AP boundary via the ciCe2 allele, the need for Gro-mediated hh repression is able to be largely abolished, with the exception of the DV boundary. Since Gro is a ubiquitous co-repressor, one has to postulate the existence of a DNA-tethering factor, which will be referred to as 'X' for the purpose of this discussion, and some process of spatial regulation of the X-Gro complex activity. The possibility that X is a form of Ci itself was tested and the answer was negative: using three different assays -- GST pulldowns, yeast two-hybrid and transfection colocalization -- no interaction between Gro and either form of Ci could be shown. Most importantly, the fact that Ci[rep] does not require Gro to repress hh in anterior cells away from the boundary supports a model where Ci and Gro repress hh independently of each other (Apidianakis, 2001).

The quantitative aspect of hh derepression in gro- clones is intriguing: clones abutting the AP boundary (type I) express the highest hh-lacZ levels, which drop gradually as clones arise further from the P compartment. This might reflect the fact that Ci[rep]-dependent repression gradually increases away from the boundary, and this is independent of gro. This interpretation assumes that basal (unrepressed) hh transcription in the A compartment would be high and subject to the dual repressors (Ci and X-Gro). Alternatively, basal hh transcription could be low, but, in addition to the repression control, hh could display a positive response to Hh signaling at the AP boundary. The latter model is consistent with the fact that in ci- cells, basal hh expression appears to be low. It also agrees with the behavior of large type I gro- clones in the present study. In these clones, high levels of hh-lacZ could be observed throughout the clone, even at a distance from the AP boundary. This could be accounted for by Hh signaling, which, having risen over some threshold owing to hh derepression, further stimulates hh transcription to a high level. This effect would spread to the edge of the clone, beyond which activation of the X-Gro repressor would silence hh transcription. The putative inducer of hh by Hh signaling may be Ci[act], as with all other direct Hh target genes; alternatively, it may be another factor induced by Ci[act]. The hypothesis that Ci[act] itself can activate hh transcription is not unreasonable, since hh should contain a regulatory region(s) that bind(s) Ci[rep]. Ci[act] and Ci[rep] contain the same DNA-binding domain and recent work has shown that the two forms of Ci bind the same target sites, although some enhancers may be configured in such a way as to respond preferentially to either the activator or repressor form (Apidianakis, 2001).

For the sake of simplicity, the existence of a low level ubiquitous activator of hh (basal levels) with a stronger activator located in P cells is postulated to account for the high levels of hh expression in P cells. In A cells that do not receive the Hh signal, the basal activity of hh is repressed by Ci[rep] and gro is not required. In A cells close to the Hh source, the basal transcription of hh would be enhanced by positive autoregulation; however, the presence of the repressive X-Gro complex does not allow this activation to take place. Implicit in this model is that X is itself activated by Hh (e.g. transcriptionally induced via Ci[act]), so that it only functions in Hh-receiving cells. In addition X production/activity should be spatially limited to the A compartment (e.g. repressed by En), since ectopic expression of full-length ci in the posterior cannot induce X-Gro activity to repress endogenous hh. According to this model, ci- clones close to the AP boundary express basal hh levels, since they lack both the X-Gro repressor (no activation of X in the absence of Ci[act]) and the activator of hh transcription (Ci[act] itself or a downstream target). By contrast, gro- clones in the same region only lack the repressive X-Gro complex and thus actively transcribe hh in response to Ci[act]; the high levels of hh produced are sufficient to initiate Hh signaling, which can propagate this effect of hh derepression throughout the clone (Apidianakis, 2001).

gro- clones near the DV boundary behave somewhat aberrantly. hh-lacZ derepression there is more efficient, observable in further anteriorly arising clones, compared with equivalent clones away from the DV boundary -- it even occurs in the presence of increased Ci[rep]. Although the mechanism remains to be discovered, one way to account for this special behavior, without invoking additional regulators, is that Ci[rep] is less active near the DV boundary and/or Ci[act] is more active, and this modulation of Ci activity in favor of the activator form allows high level hh expression at a greater distance from the Hh source and even in the ciCe2/+ background. Interestingly, ci- clones show little or no hh-lacZ derepression at the DV boundary, consistent with Gro, rather than Ci[rep], being the major hh repressor there (Apidianakis, 2001).

The model put forward here is perhaps the simplest, but by no means the only one that fits the existing data. For example, Gro might interact with Ci[act] itself, switching it from an activator into a repressor, given the right enhancer context, much like the effect Gro has on other activators, such as Dorsal. This interaction may be weak and/or require additional factors, accounting for the inability to detect it. To resolve the mechanism of hh repression at the AP boundary will necessitate detailed molecular dissection of the hh regulatory regions and characterization of relevant trans acting factors. Whatever the mechanism, it appears that a Gro-containing complex is deployed in the wing to block the spread of hh expression anteriorly from the AP compartment boundary. This should ensure a spatially fixed organizer (dpp expression stripe), in contrast to a moving one, as found in the fly retina (Apidianakis, 2001).

Drosophila segment borders result from unilateral repression of hedgehog activity by wingless signaling: Cubitus interruptus and Pangolin, function as activator and symmetry-breaking repressor of stripe expression, respectively

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).

Subfragments of the sr1.9 element lacking sr239 failed to activate discernible gene expression, whereas sr239 directs sr1.9-like gene expression in the row of cells posterior to the en domain. Moreover, sr239-dependent gene activation could be achieved upon ectopic CiZn/C (the active form of Ci) expression within the en domain. Thus, the Ci binding site-containing sr239 element is both necessary and sufficient to activate gene expression in segment border precursor cells, and it is sufficient to mediate gene activation in response to dominant active Ci. It was next asked whether the two Ci binding sites within the sr239 element are necessary to mediate Hh-dependent gene activation. For this experiment, sr239 variants lacking either one or both Ci binding sites were generated. sr239 variants lacking only one functional Ci binding site can mediate gene expression in the correct spatial pattern, but the level of expression is strongly reduced. In contrast, lack of both Ci binding sites abolished Hh/Ci-dependent gene activation completely. In summary, these findings establish that Ci activates gene expression in segment border cells. They confirm, by direct means, that Hh signaling acts not only anteriorly and across the PS boundary to maintain Wg activity, but functions in a symmetric fashion and thereby determines the position of the segment border within the PS (Piepenburg, 2000).

Since Hh signaling appears to be symmetric, it was of interest to know why the sr1.9 element fails to mediate gene activation in the row of cells anterior to the en domain. An explanation for this phenomenon would be that signaling by Wg causes region-specific repression, preventing gene activation by Hh-dependent Ci. To test this proposal, Wg was ectopically expressed in the ptc domain and the change of sr1.9-driven gene expression was examined in such embryos. Furthermore, the sr1.9-mediated gene activation was analyzed in embryos mutant for lines (lin), sloppy paired (slp), and naked (nkd). Each of these mutant embryos express en, but the wg pattern is altered (Piepenburg, 2000 and references therein).

sr1.9-mediated gene expression was abolished in response to ectopic Wg activity. The same effect was observed in nkd mutant embryos where Wg is expressed at each side of an enlarged en domain. Conversely, in slp and lin mutants where Wg activity is not maintained, sr1.9-mediated gene expression is found in two rows of cells, one on each side of the en domain. Furthermore, sr1.9-mediated gene expression was also observed in cells anterior to the region of en expression in embryos in which late Wg activity was abolished due to a temperature-sensitive wg mutation. This establishes that the repression of Hh action in the cells anterior to the wild-type en domain is dependent on Wg activity (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).

Ci regulates proliferation of ovarian somatic cells as well as specification of polar cells

Throughout Drosophila oogenesis, specialized somatic follicle cells perform crucial functions in egg chamber formation and in signaling between somatic and germline cells. In the ovary, at least three types of somatic follicle cells, polar cells, stalk cells and main body epithelial follicle cells, can be distinguished when egg chambers bud from the germarium. Although specification of these three somatic cell types is important for normal oogenesis and subsequent embryogenesis, the molecular basis for establishment of their cell fates is not completely understood. Studies reveal the gene eyes absent (eya) to be a key repressor of polar cell fate. Eya is a nuclear protein that is normally excluded from polar and stalk cells, and the absence of Eya is sufficient to cause epithelial follicle cells to develop as polar cells. Furthermore, ectopic expression of Eya is capable of suppressing normal polar cell fate and compromising the normal functions of polar cells, such as promotion of border cell migration. Finally, it has been shown that ectopic Hedgehog signaling, which is known to cause ectopic polar cell formation, does so by repressing eya expression in epithelial follicle cells (Bai, 2002).

Drosophila oogenesis provides an excellent system with which to study the mechanisms underlying specification of different cell fates. The Drosophila ovary is made up of germline cells and somatic follicle cells. Germline and somatic stem cells can be found at the anterior end of the ovary in a structure called the germarium. Germline stem cells divide asymmetrically and produce cystoblasts, which undergo four rounds of incomplete cell division and give rise to 16-cell germline cysts. One of the cyst cells becomes the oocyte and the remaining 15 cells differentiate as nurse cells. In the germarium, somatic follicle cells surround the 16-cell cysts. As the nascent egg chamber buds off from the germarium, at least three types of somatic cells can be distinguished by their morphologies and locations: polar cells, stalk cells and epithelial follicle cells. Polar cells are pairs of specialized follicle cells at each pole of the egg chamber, whereas the five to eight stalk cells separate adjacent egg chambers. Stalk and polar cells may descend from a common precursor. They differentiate and cease division soon after egg chambers form. The remaining somatic follicle cells, referred to here as epithelial follicle cells, proliferate until stage 6 of oogenesis and form a continuous epithelium around the sixteen germ cells. Subsequently, further differentiation of epithelial follicle cells occurs (Bai, 2002).

Ectopic Hh signaling produces numerous effects in the Drosophila ovary, which include regulating proliferation of somatic cells as well as specification of polar cells. Both of these effects appear to be achieved through the cell autonomous action of Ci. This raises the question of how different effects are elicited by the same signal. The data presented here indicate that ectopic Hh activates polar cell fate by repressing eya expression, the function of which is required to repress polar cell fate. Since loss of eya does not mimic ectopic Hedgehog in causing extra proliferation, it is not yet clear what factors act downstream of ectopic Hh to affect proliferation (Bai, 2002).

The relationship between Eya and Ci is not a simple linear one. Although Eya expression is repressed by CiAC, mutations in eya also alter the balance between CiAC and CiR, without affecting overall ci expression. CiAC is upregulated in eya mutant follicle cells. In addition, some of the ectopic polar cells in eya mosaic egg chambers express ptc-lacZ, which is an indicator for activation of Ci. Thus, there appears to be mutual repression between CiAC and Eya. One place in the mammalian embryo where a similar relationship between Ci and Eya might exist is in patterning the eye field. Hh is normally expressed at the midline where it represses eye development. In the absence of Hh, a single cyclopic eye forms at the midline. The three mammalian homologs of Eya are all expressed in the eye primordium and therefore it may be that the antagonism between Hh and Eya revealed in this study is also employed in the mammalian embryo to repress midline eye development (Bai, 2002).

Cubitus interruptus regulates cell cycle genes

Although mutations that activate the Hedgehog (Hh) signaling pathway have been linked to several types of cancer, the molecular and cellular basis of Hh's ability to induce tumor formation is not well understood. A mutation in patched (ptc), an inhibitor of Hh signaling, was identified in a genetic screen for regulators of the Retinoblastoma (Rb) pathway in Drosophila. Hh signaling promotes transcription of Cyclin E and Cyclin D, two inhibitors of Rb, and principal regulators of the cell cycle during development in Drosophila. Upregulation of Cyclin E expression, accomplished through binding of Cubitus interruptus (Ci) to the Cyclin E promoter, mediates the ability of Hh to induce DNA replication. Upregulation of Cyclin D expression by Hh mediates the distinct ability of Hh to promote cellular growth. The discovery of a direct connection between Hh signaling and principal cell-cycle regulators provides insight into the mechanism by which deregulated Hh signaling promotes tumor formation (Duman-Scheel, 2002).

During eye development in Drosophila, initiation of neural differentiation, marked by an indentation referred to as the morphogenetic furrow, begins at the posterior end of the disc and passes anteriorly. Cells within the furrow arrest in G1 phase before differentiating. Cells located just posterior to the furrow exit G1 arrest and enter a synchronous S phase referred to as the second mitotic wave. Overexpression of the Drosophila Retinoblastoma-family gene (Rbf), an inhibitor of the S phase promoting transcription factor E2F, produces a 'rough' adult eye phenotype, characterized by loss of bristles and fusion of ommatidia. This phenotype results from delay of S phase progression in cells of the second mitotic wave as a consequence of inhibited E2F target gene expression. Loss of one copy of the ptc gene suppresses this rough eye phenotype and restores E2F target gene expression. The observed genetic interaction between Rbf and ptc suggests that the Hh signaling pathway might regulate the cell cycle during eye development (Duman-Scheel, 2002).

Hh is secreted from differentiating neurons located just posterior to cells entering S phase in the second mitotic wave. This expression pattern is consistent with the idea that reception of the Hh signal might be required for S phase entry in the second mitotic wave. To test this hypothesis, the effect of blocking Hh signaling during eye development was assessed. Cells with a mutated smoothened (smo) gene cannot respond to the Hh signal and fail to enter S phase in the second mitotic wave. Conversely, when Ci, the transcription factor that mediates Hh signaling, is overexpressed in the furrow, cells normally arrested in G1 enter S phase. Ectopic expression of Ci can also promote S phase in G1-arrested cells located in the wing margin and in the brain. Thus, Ci can induce S phase in a variety of tissues (Duman-Scheel, 2002).

Cyclin D, a G1/S cyclin, promotes S phase by inhibiting Rb. During eye development, Cyclin D is expressed in the furrow, and its highest level of expression overlaps with Ci expression. This expression pattern is consistent with the idea that Ci protein, which is stabilized in response to reception of the Hh signal from posterior neurons, promotes expression of Cyclin D in the eye. In support of this idea, Cyclin D levels are drastically reduced in smo mutant clones that extend through the furrow. Conversely, overexpression of Ci induces high levels of Cyclin D transcript and protein expression in the furrow and in cells immediately surrounding the furrow. In the eye, the ability of Ci to induce high levels of Cyclin D expression is limited to the vicinity of the furrow, the only region where it is capable of inducing S phase. Overexpression of Ci also induces high levels of Cyclin D transcript and protein expression when expressed ectopically in the wing disc. Thus, in addition to promoting S phase, Ci induces expression of Cyclin D in both the eye and wing (Duman-Scheel, 2002).

The ability of Hh signaling to induce expression of Cyclin D may explain why increased Hh signaling suppresses phenotypes associated with RBF overexpression. In support of this idea, overexpression of Ci, which induces Cyclin D expression, also induces ectopic expression of PCNA, an E2F target gene, in both the furrow and in G1-arrested cells located in the wing margin. Coexpression with Ci of RBF-280, a constitutively active form of RBF that cannot be regulated by Cyclin D or Cyclin E, blocks the ability of Ci to induce ectopic PCNA expression in the furrow and wing margin. However, although RBF-280 can block the ability of Ci to induce E2F target gene expression, it does not block the ability of Ci to promote S phase in the eye. These results indicate that although Hh signaling induces E2F target gene expression, it must also be capable of inducing S phase independently of E2F (Duman-Scheel, 2002).

A second G1/S cyclin, Cyclin E, is a principal regulator of S phase during Drosophila development. Although Cyclin E is an inhibitor of Rb, it also has additional Rb/E2F-independent cell-cycle roles. The initiation of high levels of Cyclin E expression in cells of the second mitotic wave that is located just anterior to neurons secreting Hh protein, is consistent with the idea that Hh signaling may regulate Cyclin E expression in the eye. Indeed, loss of smo results in reduction of Cyclin E levels in cells entering the second mitotic wave. Conversely, when Ci is overexpressed in furrow cells, high levels of Cyclin E transcript and protein can be detected within these cells. In the eye, the ability of Ci to induce high levels of Cyclin E expression is limited to the furrow region, where it is capable of inducing S phase. Overexpression of Ci is also capable of inducing high levels of Cyclin E transcript and protein expression in the wing. Although Cyclin E is an E2F target gene, the ability of Ci to promote Cyclin E expression in the presence of RBF-280 indicates that Hh can induce Cyclin E expression independently of E2F. Therefore, in addition to promoting expression of Cyclin D and activating E2F, Hh signaling also promotes expression of Cyclin E independently of E2F. The ability of the Cyclin E-dependent kinase inhibitor Dacapo (Dap) to inhibit the ability of Ci to induce S phase in the furrow and wing margin indicates that Cyclin E is the principal mediator of the ability of Hh to promote S phase (Duman-Scheel, 2002).

To investigate the mechanism by which Hh signaling induces Cyclin E transcription, the Cyclin E promoter was examined. Several sequences with homology to the consensus Ci-binding site were identified within the 5' regulatory region of Cyclin E. Ci was found to bind to three Ci-binding sites (A, B and C in gel shift competition experiments). To examine whether this interaction occurs in vivo, chromatin immunoprecipitation (ChIP) experiments were carried out. These assays demonstrate that Ci-binding sites A-C are occupied by Ci protein in vivo. Several lines of evidence indicate that the observed ability of Ci to bind to the Cyclin E promoter is important for regulation of Cyclin E expression in the developing eye. First, normal upregulation of Cyclin E expression in the second mitotic wave is disrupted in Cyclin EJP mutant flies, which bear a P element inserted adjacent to Ci-binding sites A-C. Also, flies carrying the 16.4 Cyclin E lacZ13 reporter, which contains all three Ci-binding sites, show upregulation of ß-galactosidase (ß-gal) expression in the second mitotic wave; this pattern resembles the endogenous pattern of Cyclin E expression. By contrast, upregulated ß-gal levels in the second mitotic wave are not observed in flies carrying reporter 13.2 Cyclin E lacZ13, which lacks Ci-binding sites A and B. Furthermore, overexpression of Ci in the furrow can drive ectopic ß-gal expression from reporter 16.4, but not from reporter 13.2. These experiments suggest that the presence of sites A and B is required for normal Hh-mediated upregulation of Cyclin E expression in the second mitotic wave. Taken together, these results provide strong evidence that Hh signaling promotes S phase through direct induction of Cyclin E expression by the transcription factor Ci (Duman-Scheel, 2002).

Cell growth is thought to be regulated independently of cell proliferation. For example, overexpression of several well-characterized cell-cycle regulators induces cell proliferation but fails to stimulate growth (defined as the accumulation of mass). In contrast, Cyclin D induces both cell proliferation and promotes the accumulation of mass. Thus, it seems likely that Hh, which induces expression of Cyclin D, may also have a distinct function in regulating cell growth. To test how the modulation of Hh signaling influences growth the effects of overexpression of Ptc or Ci in clones of undifferentiated wing disc cells werre examined. Estimation of clone size was used as a measure of growth. Ptc overexpression clones are significantly smaller than control clones and cover only 65% of the area covered by control clones. In contrast, Ci overexpression clones are significantly larger than control clones and cover 143% of the area covered by control clones. These data indicate that, in addition to promoting S phase, Hh signaling has a distinct function in regulating cell growth. The observed ability of Hh signaling to promote cellular growth correlates with previous experiments indicating that Ptc overexpression results in reduced tissue growth (Duman-Scheel, 2002).

It is likely that Cyclin D, which is upregulated upon overexpression of Ci in the wing , might mediate the ability of Hh to promote growth. Consistent with this hypothesis, overexpression of Ci, like overexpression of Cyclin D-Cyclin-dependent kinase 4 (Cdk4), induces growth and accelerates cell proliferation rates without affecting individual cell size or overall cell-cycle phasing. Likewise, although inhibiting the Hh pathway by overexpressing Ptc decreases growth and decelerates cell proliferation rates, it does not affect individual cell size or cell cycle phasing (Duman-Scheel, 2002).

The ability of Cyclin D-Cdk4 to mediate induction of growth by Hh was examined in several ways. First, the ability of Cyclin D-Cdk4 to rescue the inhibitory effects of Ptc on growth was examined. The average size of clones expressing Ptc and Cyclin D-Cdk4 is significantly larger than the size of Ptc overexpression clones and is comparable to the size of clones expressing CyclinD-Cdk4 alone. Thus, overexpression of CyclinD-Cdk4 can suppress the inhibition of growth by Ptc. Furthermore, although clones of cells lacking Cdk4 (the only apparent kinase subunit for Drosophila Cyclin D) grow more slowly than wild-type cells, overexpression of Ptc does not significantly reduce the size of clones induced in a Cdk4 mutant background. Also, overexpression of Ci cannot stimulate growth in a Cdk4 mutant background. These results indicate that Cyclin D-Cdk4 is the principal growth-regulating target of Hh signaling (Duman-Scheel, 2002).

The investigation demonstrates that Hh signaling has a distinct ability to promote cellular growth, which is mediated by Cyclin D. In addition, Hh signaling can induce proliferation during development by promoting expression of Cyclin D and Cyclin E. This study reveals a direct connection between Hh signaling and induction of Cyclin E expression, which is accomplished through binding of Ci to the Cyclin E promoter. Upregulation of murine cyclin D1, D2 and E in response to Hh signaling has been observed. It is therefore likely that the mechanism for Cyclin E induction by Hh described here is conserved in mammals. Furthermore, because both overexpression of Ptc-1 or mutation of cyclin D1 produces a small mouse phenotype, it is likely that the ability of Hh to promote cellular growth through upregulation of D-type cyclins is also conserved in mice. Thus, constitutive Hh signaling (which promotes deregulated expression of G1/S cyclins that have been associated with diverse forms of human cancer) would promote both cell proliferation and growth in tumors. In contrast, during development, cell growth and proliferation must be carefully regulated and coordinated with the processes of cell patterning and differentiation. These same processes are also regulated by Hh signaling. This delicate balance is probably maintained by tight control of the temporal and spatial expression patterns of Hh targets and the molecules that regulate them (Duman-Scheel, 2002).

Cadherin Cad99C is regulated by Ci in the wing disc

The subdivision of the Drosophila wing imaginal disc into anterior and posterior compartments requires a transcriptional response to Hedgehog signaling. However, the genes regulated by Hedgehog signal transduction that mediate the segregation of anterior and posterior cells have not been identified. The previously predicted gene Cad99C has been molecularly characterized and shown to be regulated by Hedgehog signaling. Cad99C encodes a transmembrane protein with a molecular weight of approximately 184 kDa that contains 11 cadherin repeats in its extracellular domain and a conserved type I PDZ-binding site at its C-terminus. The levels of cad99C RNA and protein are low throughout the wing imaginal disc. However, in the pouch region, these levels are elevated in a strip of anterior cells along the A/P boundary where the Hedgehog signal is transduced. Ectopic expression of Hedgehog, or the Hedgehog-regulated transcription factor Cubitus interruptus, induces high-level expression of Cad99C. Conversely, blocking Hedgehog signal transduction by either inactivating Smoothened or Cubitus interruptus reduces high-level Cad99C expression. Finally, by analyzing mutant clones of cells, it was shown that Cad99C is not essential for cell segregation at the A/P boundary. It is concluded that cad99C is a novel Hedgehog-regulated gene encoding a member of the cadherin superfamily in Drosophila (Schlichting, 2005).

The predicted Cad99C amino acid sequence contains the characteristic features common to members of the cadherin superfamily including a signal peptide, cadherin domains flanked by Ca++-binding sites, and a transmembrane domain, defining Cad99C as a member of this superfamily. A β-catenin binding site, which is present in the cytoplasmic domain of type-I and type-II cadherins providing a link to the actin cytoskeleton, was not identified. However, a conserved type I PDZ-binding sequence was identified at the C-terminus of Cad99C. PDZ domain-containing proteins commonly act as linkers that facilitate the assembly of large molecular complexes within cells, suggesting that Cad99C physically interacts with a cytosolic protein complex. Only few other cadherins have been reported to contain C-terminal PDZ-binding sequences. Cadherin 23, for example, a vertebrate cadherin lacking the consensus R1 and R2 β-catenin binding sites, associates with its C-terminal PDZ-binding site with the PDZ domain-containing protein harmonin b. Interestingly, harmonin b has been shown to be able to bundle actin filaments, providing a potential link between cadherin 23 and the actin cytoskeleton independent of β-catenin. It is therefore conceivable that Cad99C interacts with a PDZ domain-containing protein that provides a link to the actin cytoskeleton independent of β-catenin. The PDZ-binding sequence of Cad99C is adjacent to and overlapping with two predicted Ser/Thr kinase phosphorylation sites. The interaction between a PDZ-binding peptide and a PDZ domain can be disrupted by phosphorylation of the PDZ-binding sequence by Ser/Thr kinases, suggesting that the potential binding of Cad99C to a PDZ domain-containing protein could be regulated by phosphorylation (Schlichting, 2005).

Cad99C is expressed at low levels throughout the wing, haltere, and leg imaginal discs, whereas elevated levels of Cad99C expression are confined to a strip of cells along the A/P boundary of the wing imaginal disc pouch that is known to respond to the Hh signal. Even though anterior cells along the A/P boundary of haltere and leg imaginal discs as well as cells outside the pouch region of wing imaginal discs also respond to the Hh signal, no elevated level of Cad99C was observed in these cells, indicating that cad99C is a region-specific Hh target gene. The Cad99C protein profile resembles cad99C RNA levels, indicating that the elevated expression of Cad99C is mainly due to transcriptional and not translational or posttranslational regulation. High-level Cad99C expression was reduced to the low level present in cells far away from the A/P boundary in clones of cells lacking Hh signal transduction due to mutations in either smo or ci. Conversely, ectopic expression of either Hh or Ci was sufficient to increase Cad99C expression in the wing imaginal disc pouch, indicating that high-level cad99C expression is controlled by Ci-mediated Hh signaling (Schlichting, 2005).

Different Hh-regulated genes respond differently to Ci[act] and Ci[rep]. For example, the expression of dpp is regulated both by Ci[act] and Ci[rep], whereas hh and ptc only respond to one form of Ci, Ci[rep] or Ci[act], respectively. Like ptc, cad99c appears to respond exclusively to Ci[act]. This is inferred from five observations: (1) ectopic expression in posterior cells of Ci, which under the influence of Hh is converted to Ci[act], induces high-level cad99C expression; (2) misexpression of a constitutively active form of Ci, CiPKA4, also induces high levels of cad99C expression; (3) ci null mutant clones in the anterior compartment close to the A/P boundary, where Ci[act] is the predominant form of Ci, fail to upregulate cad99C expression; (4) expression of a constitutive repressor form of Ci, CiCell, does not reduce the low-level expression of cad99C; (5) ci null mutant clones in the anterior compartment away from the A/P boundary, where Ci[rep] is the prevailing form of Ci, show no increase in the expression of cad99C. Taken together, it is concluded that cad99C expression is regulated by Ci[act] and not Ci[rep] (Schlichting, 2005).

The segregation of cells at compartment boundaries is thought to depend on the differential adhesiveness (affinity) of cells on both sides of the compartment boundaries. Based on thermodynamic considerations, it has been proposed that cells will maximize the total strength of their adhesive interactions with neighboring cells by replacing weak cell–cell interactions with stronger ones. Cells with strong adhesive interactions will thus associate preferentially with one another and will segregate from less avidly adhering cells. As predicted by this model, cells expressing different levels of the same adhesion molecule segregate from one another. However, few adhesion molecules have been identified that can promote the differential adhesiveness of cells at compartment boundaries (Schlichting, 2005).

The maintenance of the A/P boundary in the developing Drosophila wing requires Ci-mediated Hh signal transduction in anterior cells. This suggests that Hh signaling may regulate the transcription of one or more genes that in turn affect the adhesiveness of anterior cells. Members of the cadherin superfamily are known to mediate adhesion between cells and several cadherins have been shown to be involved in cell segregation. Even though most cadherins implicated so far in cell segregation contain cytoplasmic β-catenin binding sites, which are absent in Cad99C, several cadherins lacking β-catenin binding sites have also been shown to mediate cell segregation. The discovery of a gene that is both regulated by Hh signaling and encodes for a cadherin, therefore, provides an attractive candidate for mediating the segregation of anterior and posterior cells. However, cad99C expression is not elevated in cells along the A/P boundary of haltere and leg imaginal discs or outside the pouch region of wing imaginal discs, indicating that if the elevated expression of Cad99C were important for cell segregation, this could not be a general mechanism for segregating anterior and posterior cells. However, since wing imaginal disc pouch cells differ in their expression profile from wing imaginal disc cells outside of the pouch, it is not inconceivable that different molecules could operate to segregate cells at the A/P boundary in different regions of the wing imaginal disc or in different imaginal discs (Schlichting, 2005).

A mutant allele of cad99C, termed cad99C57A, was generated in order to test whether Cad99C is required to segregate anterior and posterior cells. cad99C57A appears to be a null allele of cad99C based on four criteria: (1) sequencing of the genomic DNA revealed that the predicted promoter region, the transcriptional start site, and the coding sequence for the first 101 amino acid residues were deleted; (2) an RNA probe recognizing the 3′ region of the cad99C transcript, outside of the deletion present in cad99C57A, does not show detectable staining in wing imaginal discs from homozygous cad99C57A mutant larvae, indicating that the cad99C transcript levels are highly reduced; (3) an antibody directed to the C-terminus of Cad99C does not recognize a protein of the predicted size for Cad99C in extracts from wing imaginal discs of homozygous cad99C57A mutant larvae; (4) Cad99C immunoreactivity is highly reduced in homozygous cad99C57A mutant clones within wing imaginal discs (Schlichting, 2005).

The requirement for Cad99C to maintain the segregation of anterior and posterior wing imaginal disc cells was tested by clonal analysis. Using this assay, Cad99C was found not to be essential for maintaining the normal segregation of anterior and posterior cells. This result can be explained in several ways. (1) Cad99C does not play any role in the segregation of anterior and posterior cells. The Hh-dependent increase in expression of Cad99C may either be irrelevant for the function of Cad99C or might reflect an unrelated function. For example, Hh signaling is required for the patterning of the longitudinal wing veins L3 and L4. It is thus conceivable that Cad99C may play a role in this aspect of Hh signaling. However, vein patterning appeared to be normal in wings from homozygous cad99C57A mutants. (2) The activity of Cad99C in mediating the segregation of anterior and posterior cells is redundant with the activity of one or several of the remaining 16 cadherins in Drosophila. Different, partially redundant mechanisms contribute to the segregation of anterior and posterior cells. For example, one mechanism might be Cad99C-dependent whereas additional mechanisms may rely on cell surface proteins unrelated to cadherins or on cytoskeletal components (Schlichting, 2005).

The identification of cad99C as an Hh-regulated gene provides a starting point to investigate a cell biological mechanism used by Hh signaling to control the development of the Drosophila wing. It also provides a further step towards the functional characterization of all remaining members of the cadherin superfamily present in Drosophila that have so far only been predicted based on the genomic sequence (Schlichting, 2005).

Ci targets knot in the wing disc

The regulation of development by Hox proteins is important in the evolution of animal morphology, but how the regulatory sequences of Hox-regulated target genes function and evolve is unclear. To understand the regulatory organization and evolution of a Hox target gene, a wing-specific cis-regulatory element was identified controlling the knot gene, which is expressed in the developing Drosophila wing but not the haltere. This regulatory element contains a single binding site that is crucial for activation by the transcription factor Cubitus interruptus (Ci), and a cluster of binding sites for repression by the Hox protein Ultrabithorax (Ubx). The negative and positive control regions are physically separable, demonstrating that Ubx does not repress by competing for occupancy of Ci-binding sites. Although knot expression is conserved among Drosophila species, this cluster of Ubx binding sites is not. The knot wing cis-regulatory element was isolated from D. pseudoobscura, which contains a cluster of UBX-binding sites that is not homologous to the functionally defined D. melanogaster cluster. It is, however, homologous to a second D. melanogaster region containing a cluster of UBX sites that can also function as a repressor element. Thus, the knot regulatory region in D. melanogaster has two apparently functionally redundant blocks of sequences for repression by UBX, both of which are widely separated from activator sequences. This redundancy suggests that the complete evolutionary unit of regulatory control is larger than the minimal experimentally defined control element. The span of regulatory sequences upon which selection acts may, in general, be more expansive and less modular than functional studies of these elements have previously indicated (Hersh, 2005).

The knot gene is expressed in the developing Drosophila wing imaginal disc at the anteroposterior compartment boundary, but is not expressed in the haltere imaginal disc. Furthermore, knot expression is genetically downstream of Ubx; overexpression of Ubx in clones in the wing causes cell-autonomous loss of knot expression. Because these features make knot a candidate for direct regulation by Ubx, attempts were made to identify the regulatory element that controls knot expression in the wing (Hersh, 2005).

Expression of the knot gene is dependent on Hedgehog (Hh) activity, and overexpression of Hh can trigger ectopic knot expression in the wing. The transcriptional effector of Hh signaling is the Cubitus interruptus (Ci) protein. Ci is a zinc-finger transcription factor of the Gli family, and binds a 9 bp consensus sequence TGGG(T/A)GGTC. In the 1.3 kb knMel701-1991 fragment, three potential Ci-binding sites were identified that matched at least seven out of nine consensus residues and that are conserved in D. pseudoobscura. Two additional potential sites were present, but were not conserved in D. pseudoobscura. The three conserved binding sites were independently mutagenized, converting a crucial guanine to an adenine, and the mutagenized element was re-introduced into flies. Changes at two of the three candidate sites had no effect on reporter gene expression, whereas the mutation of site Ci1680 almost completely abolishes reporter expression. Mutation of all three sites did not have a more severe effect than mutation of Ci1680 alone. These results indicate that activation of the wing-specific enhancer element by Hh signaling is dependent primarily on a single Ci-binding site at position 1680 in the knMel701-1991 element (Hersh, 2005).

Although Ubx site 1 is located only 4 bp from the Ci-binding site, it is still present in the knMel701-1835 construct that is derepressed in the haltere, so this site alone is not sufficient to mediate repression by Ubx. To determine whether this site is necessary for repression by Ubx, Ubx site 1 alone (knMel701-1991Ubx1KO) was mutated and no derepression of reporter gene expression in the haltere was observed. Therefore, Ubx Site 1, unlike individual Ubx-binding sites in the spalt enhancer, does not appear to contribute significantly to repression of this element by Ubx. Of the other regions protected by Ubx, the largest spans six TAAT core sequences and ~24 bp of sequence, and is located ~250 bp from the Ci binding site. Therefore, the DNA sequences necessary for repression in the haltere appear to be comprised of multiple, functional Ubx-binding sites that do not overlap with the activating Ci-binding site. This organization suggests that Ubx does not repress knot in the haltere by competing for activator binding sites (Hersh, 2005).

Thus, a wing-specific cis-regulatory element was identified for the gene knot. This regulatory element is activated in the wing by direct input from Ci and is repressed in the haltere by direct input from Ubx. The regulatory sequences governing activation and repression are physically separable, and the repression element was found not to be shared with D. pseudoobscura. A distinct functional repression element was identified in D. pseudoobscura that is shared with D. melanogaster, indicating that the entire knot wing regulatory region in D. melanogaster contains two apparently redundant repressor elements. One element appears to have been acquired in the course of the evolution of the D. melanogaster lineage. These results suggest that complete functional cis-regulatory elements, the units of function that selection is operating upon, may be larger and more diffuse than the minimal functional sequences typically defined by molecular dissection (Hersh, 2005).

Cubitus interruptus targets in the leg

Many of the genes that pattern Drosophila are expressed throughout development and specify diverse cell types by creating unique local environments that establish the expression of locally acting genes. This process is exemplified by the patterning of leg microchaete rows. hairy (h) is expressed in a spatially restricted manner in the leg imaginal disc and functions to position adult leg bristle rows by negatively regulating the proneural gene achaete, which specifies sensory cell fates. While much is known about the events that partition the leg imaginal disc and about sensory cell differentiation, the mechanisms that refine early patterning events to the level of individual cell fate specification are not well understood. In the third instar leg imaginal disc, h is expressed along both the D/V and A/P axes. D/V axis expression appears as a single stripe in the anterior compartment of the disc immediately adjacent to the A/P compartment boundary. A/P axis expression appears as two wedge-shaped blocks in the distal leg segments on either side of the A/P compartment boundary. After disc eversion, the D/V axis stripe forms two of the four longitudinal leg stripes. The A/P axis expression forms five circumferential stripes at the first through fifth tarsal segments. The remaining two longitudinal stripes will be positioned along the A/P axis, intersecting the D/V axis stripes at the distal tip of the leg. These stripes do not appear until 2-3 hours after puparium formation (APF). This paper concerns itself with Hairy expression in the D/V axis (Hays, 1999).

The expression pattern of the D/V axis h stripe (D/V-h) is highly reminiscent of the patterning of known Hh target genes; therefore, constituents of the Hh signal transduction pathway were examined as potential regulators of the stripe. Hh is secreted from the posterior compartment of imaginal discs and influences gene expression in anterior compartment cells. Ci, a zinc finger transcription factor, is expressed throughout the anterior compartment and is thought to mediate Hh signaling by direct transcriptional regulation of Hh target genes. The region of high-level, full-length Ci overlaps with D/V-h, making Ci a strong candidate for regulation of this h stripe. The influence of Hh on h expression was investigated by generating somatic clones which lack functional Smoothened (Smo). Within smo clones, both dorsally and ventrally, D/V-h expression is lost in a cell autonomous manner, implicating Hh signaling in D/V-h regulation. This analysis was extended to Ci, the only known transcriptional mediator of Hh signaling, by assaying the expression of h in legs misexpressing full-length Ci. In the leg disc, the 30A-GAL4 insertion is expressed dorsally in cells of the presumptive femur, in the distal tip of the leg, and in a central ring corresponding to the fifth tarsal segment. Misexpression of a ci transgene under control of the 30A-GAL4 driver results in ectopic expression of h throughout the 30A expression domain, suggesting that ci can serve as a positive regulator of h (Hays, 1999).

In order to independently assay the response of D/V-h to Ci, the leg-specific enhancer elements which govern the expression of D/V-h were isolated and cloned into a lacZ reporter vector. A 9 kb genomic fragment 30-40 kb 3' of the h transcription start site contains sequences that direct beta-gal expression in the D/V-h stripe domain. This reporter construct is referred to as D/V-h- lacZ. The D/V-h-lacZ stripe lies in the anterior compartment of the disc adjacent to the compartment boundary and superimposes with endogenous H protein. As with endogenous D/V-h, D/V-h-lacZ expression is lost in smo mutant clones, suggesting that this enhancer element is a target of Hh signaling. Misexpression of ci with the 30A-GAL4 driver results in ectopic expression of D/V-h-lacZ, throughout the 30A domain, though the pattern differs somewhat from that seen with endogenous H. This confirms the specific activation of D/V-h expression by exogenously supplied Ci. In addition to ectopic activation by Ci, a dominant-negative form of ci impedes expression of D/V-h (Hays, 1999).

The D/V-h enhancer sequences are separable into discrete dorsal and ventral components, referred to as D-h and V-h, respectively. Both of these elements are responsive to Ci. The separability of these enhancers suggests that D/V-h expression is regulated by dorsal- and ventral-specific factors, such as Dpp and Wg, and not by ci alone. Dpp and Wg are known to specify dorsal and ventral leg fates, respectively, and have been shown to antagonize each other’s function to maintain dorsal and ventral leg territories. In the absence of one signaling molecule, expression of the other and its target genes expands, resulting in the duplication of dorsal or ventral leg structures (Hays, 1999).

To assess the roles of Dpp and Wg in the regulation of the D-h and V-h enhancer elements, the expression of D-h-lacZ and V-h-lacZ was assayed in legs that were mutant for either dpp or wg. Expression from D-h-lacZ is reduced in leg discs that are mutant for dpp, and is expanded to produce a full D/V axis stripe in discs that are mutant for wg. Conversely, V-h-lacZ expression is severely reduced in wg mutant discs, and duplicated in dpp mutant discs. These findings are in keeping with what has been demonstrated for other Dpp and Wg target genes and suggest that the D-h and V-h enhancers are targets of Dpp and Wg signaling, respectively. The roles of Dpp and Wg in D/V-h regulation were further examined by making somatic clones lacking components of the Dpp and Wg signaling pathways. Given the antagonism between Dpp and Wg in the leg, it was necessary to analyze clones mutant for a component of each pathway. D-h expression was examined in clones mutant for wg and Mothers against dpp (Mad). Mad is a downstream effector in the Dpp signaling pathway that has been shown to bind DNA and transcriptionally regulate some Dpp target genes directly. Dorsal Mad;wg clones that intersect the D-h stripe show loss of h expression, except for variable low level expression in a single row of cells immediately adjacent to the A/P boundary. It can reasonably be concluded that loss of D-h results from the loss of Mad, since there is no duplicated Wg in these clones. These results not only support the finding that D-h is a target of Dpp signaling, it identifies Mad as a potential transcriptional regulator of D-h. Thus it is proposed that D/V-h expression is regulated in a non-linear pathway in which Ci plays a dual role. In addition to serving as an upstream activator of Dpp and Wg, Ci acts combinatorially with them to activate D/V-h expression (Hays, 1999).

Opposing inputs by Hedgehog, acting through Ci, and Brinker define a stripe of hairy expression in the Drosophila leg imaginal disc

The sensory organs of the Drosophila adult leg provide a simple model system with which to investigate pattern-forming mechanisms. In the leg, a group of small mechanosensory bristles is organized into a series of longitudinal rows, a pattern that depends on periodic expression of the hairy gene and the proneural genes achaete and scute. Expression of ac in longitudinal stripes in prepupal leg discs defines the positions of the mechanosensory bristle rows. The ac/sc expression domains are delimited by the Hairy repressor, which is itself periodically expressed. In order to gain insight into the molecular mechanisms involved in leg sensory organ patterning, a Hedgehog (Hh)- and Decapentaplegic (Dpp)-responsive enhancer of the h gene, which directs expression of h in a narrow stripe in the dorsal leg imaginal disc (the D-h stripe) has been examined. These studies suggest that the domain of D-h expression is defined by the overlap of Hh and high-level Dpp signaling. The D-h enhancer consists of a Hh-responsive activation element (HHRE) and a repression element (REPE), which responds to the transcriptional repressor Brinker (Brk). The HHRE directs expression of h in a broad stripe along the anteroposterior (AP) compartment boundary. HHRE-directed expression is refined along the AP and dorsoventral axes by Brk1, acting through the REPE. In D-h-expressing cells, Dpp signaling is required to block Brk-mediated repression. This study elucidates a molecular mechanism for integration of the Hh and Dpp signals, and identifies a novel function for Brk as a repressor of Hh-target genes (Kwon, 2004).

The D-h and V-h stripes are regulated by separate enhancers, which map between 32-38 kb 3' to the h transcription unit. ac stripes are not expressed until 6 hours after puparium formation (APF). The flanking narrow D-h stripe is positioned a few cells anterior to the compartment boundary, allowing expression of two dorsal ac stripes in the anterior compartment. V-h, however, is expressed directly adjacent to the AP boundary so that there is only one ventral ac stripe in the anterior compartment. Expression of each h stripe in its proper register is essential for positioning of the ac stripes and consequently for sensory bristle patterning in the adult leg. Focus was placed on the mechanisms that lead to expression of the D-h stripe in its precise register near the AP boundary (Kwon, 2004).

Expression of the endogenous D-h stripe is dependent on Hh signaling. In order to identify sequences that mediate Hh responsiveness, a dissection was undertaken of the D-h enhancer. The D-h enhancer maps to a 3.4 kb BamHI/EcoRI fragment located 32 kb 3' to the h structural gene. In third instar leg imaginal discs, this fragment directs lacZ expression in a dorsally restricted AP boundary-adjacent stripe. Two subfragments of the D-h enhancer were tested for the ability to drive reporter gene expression in leg imaginal discs. A 3' 2.4 kb HindIII/EcoRI subfragment of the D-h enhancer (REPE) directs no detectable reporter gene expression in leg imaginal discs. However, the complementary 5' 1.0 kb BamHI/HindIII fragment of the D-h enhancer drives expression in a stripe that is not dorsally restricted but rather traverses the entire length of the DV axis, suggesting it responds to Hh signaling in both dorsal and ventral leg cells. To determine whether Hh signals through the BamHI/HindIII fragment of the D-h enhancer, expression from a BamHI/HindIII-GFP transgene was assayed in leg clones lacking function of Smoothened (Smo), a transmembrane protein required for transduction of the Hh signal. Somatic clones lacking smo function were generated by FLP/FRT-mediated mitotic recombination. Cell-autonomous loss of GFP expression is lost in smo clones that overlap the GFP stripe. These observations imply that Hh signals through the BamHI/HindIII fragment, and therefore, this region is referred to as the D-h-Hh response element (HHRE) (Kwon, 2004).

Since the HHRE is Hh responsive, the element for the consensus binding site of the Hh pathway transcriptional effector, Ci was sought. Two potential Ci-binding sites (Ci-1 and Ci-2) were found, each of which matches the consensus, TGGG(A/T)GGTC, in a minimum of seven out of nine sites and binds the Ci zinc-finger domain (CiZn) in a electrophoretic mobility shift assay (EMSA). To determine whether the Ci-binding sites are required for HHRE-directed expression, point mutations were introduced into the Ci-1 and 2 sites. These mutations abolish Ci binding of the HHRE in vitro. Expression directed by the HHRE with a mutation in either the Ci-1 or Ci-2 sites is drastically compromised, and there is no detectable expression from an HHRE-lacZ transgene with both Ci sites mutated. Taken together, these studies indicate that D-h expression is activated primarily by the HHRE, through which Ci acts as an essential and direct transcriptional activator (Kwon, 2004).

Endogenous D-h expression is compromised in somatic clones lacking function of Mad, the transcriptional effector of Dpp signaling, and D-h-lacZ expression is severely decreased in leg imaginal discs with reduced dpp function. Furthermore, D-h-lacZ expression is ventrally expanded in wingless (wg) mutant legs, which have strong ventral dpp expression. These findings indicate a requirement for Dpp, in addition to Hh signal, for D-h expression. The most parsimonious model to explain how h integrates positive input from the Hh and Dpp signals, is that Mad acts synergistically with Ci through the D-h enhancer to activate D-h expression. However, Dpp is instead required to block REPE-mediated repression (Kwon, 2004).

Thus, the D-h activation element, HHRE, has two consensus Ci-binding sites, which bind Ci in vitro, and are required for its activity. In addition, HHRE-GFP expression is abrogated in clones lacking function of smo, a transducer of the Hh signal. These observations suggest that Ci acts directly through the HHRE to activate D-h expression. h is one of a number of genes, including dpp, patched (ptc), knot and araucan/caup (ara/caup), that have been identified as targets of Hh signaling in imaginal discs. These genes are each expressed in a stripe along the AP compartment boundary, but curiously, stripe widths among the genes varies as does register relative to the AP boundary. This has been explained in terms of differential response of Hh-target genes to the repressor and activator forms of Ci (Ci-R and Ci-A, respectively) found in anterior compartment cells. ptc, for example, has been proposed to respond only to the maximal levels of Ci-A found in cells nearest the AP boundary, while dpp responds to lower levels of Ci-A and also to Ci-R. The broad AP boundary stripe of HHRE-directed expression suggests that the HHRE is highly responsive to Ci-A. Differential response to Ci-R and Ci-A is thought to be controlled by cis-regulatory elements outside the local context (within 100 bp) of Ci binding sites in Hh responsive enhancers. Consistent with this hypothesis, an element, the REPE, has been identified that appears to modulate the response of the HHRE to Ci-A (Kwon, 2004).

Although Ci-A is an essential and important activator, which acts directly through the HHRE, it is unlikely that Ci-A function is sufficient for HHRE activity. Several studies have suggested that signal response elements in enhancers are generally not sufficient to activate gene expression. Rather, the transcriptional effectors of signals must act cooperatively with other activators to direct robust expression of target genes. This phenomenon, which has been termed 'activator insufficiency,' presumably prevents promiscuous activation of potential target genes. It is likely then, that other sites in the HHRE are required in addition to the Ci sites for expression directed by this element. For example, since the HHRE drives reporter gene expression in the wing and antennal discs as well as the leg, it might be expected that a common factor expressed in all three discs acts through the HHRE in combination with Ci. Alternatively, the enhancer might harbor sites that respond to factors specific to each disc type (Kwon, 2004).

Together, these observations are consistent with a model in which Ci, acting through the HHRE, activates D-h expression. The domain of HHRE activity can be divided into two zones, 1 and 2. The HHRE has the potential to direct expression in both zones 1 and 2, but its activity is restricted to zone 1 by Brk and perhaps a second factor, X, which binds the CRE. In zone 2 cells, Brk would bind to the CMB and repress HHRE-directed expression. It is proposed that zone 1 is defined by the overlap of Hh and high-level Dpp signaling. Dpp promotes D-h expression by repressing brk expression in zone 1. However, the presence of Mad-binding sites in the CMB suggests the potential for a more direct role for Mad in D-h regulation, perhaps in competing with Brk for binding to the CMB, or in directly mediating repression. Confirmation of a role for the Mad sites awaits further analysis of the D-h enhancer (Kwon, 2004).

Cubitus interruptus targets in the eye

Although Hedgehog (Hh) signaling is essential for morphogenesis of the Drosophila eye, its exact link to the network of tissue-specific genes that regulate retinal determination has remained elusive. In this report, it is demonstrated that the retinal determination gene eyes absent (eya) is the crucial link between the Hedgehog signaling pathway and photoreceptor differentiation. Specifically, it is shown that the mechanism by which Hh signaling controls initiation of photoreceptor differentiation is to alleviate repression of eya and decapentaplegic (dpp) expression by the zinc-finger transcription factor Cubitus interruptus (Cirep). Furthermore, the results suggest that stabilized, full length Ci (Ciact) plays little or no role in Drosophila eye development. Moreover, while the effects of Hh are primarily concentration dependent in other tissues, hh signaling in the eye acts as a binary switch to initiate retinal morphogenesis by inducing expression of the tissue-specific factor Eya (Pappu, 2003).

Misexpression of eyeless (ey) in the wing disc causes ectopic photoreceptor differentiation only in regions where both dpp and hh signaling are normally active. The simplest explanation for this effect invokes a linear regulatory hierarchy where hh induces dpp, which in turn cooperates with ey to initiate retinal morphogenesis. While misexpression of ey and dpp together does indeed lead to synergistic photoreceptor differentiation, this occurs only in the posterior compartment of the wing disc. Notably, Hh signaling is not transduced in the posterior compartment of the wing disc due to the repression of ci by En. Furthermore, dpp and ey expression does not induce Ci expression in the posterior compartment of the wing disc. Thus, it is concluded that dpp and ey can induce Eya expression and photoreceptor differentiation in the posterior compartment of the wing disc in the absence of Hh signaling and Cirep. Misexpression of hh and ey induces robust eya expression and photoreceptor differentiation in the wing disc, but only in the anterior compartment. This result is consistent with a model in which Hh signaling normally blocks the production of Cirep and converts it into an activated form, Ciact, in the anterior compartment of the wing disc. Ciact can induce dpp expression in the anterior compartment and dpp can in turn cooperate with ey to induce robust Eya expression and photoreceptor differentiation. Consistent with this model, co-expression of hh, dpp and ey leads to Eya expression and photoreceptor differentiation in both compartments of the wing disc. Taken together, these results suggest that, in the wing disc, ey and dpp can activate eya expression only in the absence of Cirep (Pappu, 2003).

Co-expression of dpp, ey and eya using the 30A-Gal4 driver induces photoreceptor differentiation in both wing compartments, albeit with low penetrance. This effect becomes stronger and more penetrant when dpp, ey, eya and so are misexpressed in a ring around the wing pouch. These results demonstrate that providing ey, dpp and eya from an exogenous source is sufficient to bypass the requirement for Hh signaling during initiation of ectopic photoreceptor differentiation. In addition, these results implicate eya as a key target for Hh signaling during the initiation of normal retinal morphogenesis, most likely by blocking Cirep (Pappu, 2003).

The data from ectopic expression analyses in the wing disc suggest that Cirep has a major role in blocking eya expression in areas that are not exposed to Hh signaling. However, Ciact also plays an important role in patterning the anterior compartment of the wing disc. For example, adult wings that contain ci mutant clones develop with defects in the anterior compartment. In the Drosophila eye disc, ci is expressed uniformly but Ci protein expression follows a dynamic pattern. It has been proposed that in regions anterior to the furrow Ci is subject to PKA-dependent phosphorylation and SCFSlimb-dependent processing into Cirep. Cells in the MF, however, receive and transduce the Hh signal and prevent the proteolytic processing of Ci, therefore blocking production of Cirep. Furthermore, it has been proposed that cells that are posterior to the MF do not accumulate Cirep in a PKA-dependent manner. Instead, these cells use a smo- and cullin3- dependent proteolytic process leading to the complete degradation of Ci. Therefore, the role for Ci in the eye appears to be limited only to cells that are part of, and anterior to, the MF. However, these studies do not establish separate functional roles for Ciact and Cirep in the developing eye (Pappu, 2003).

Surprisingly, Eya expression and photoreceptor differentiation are not perturbed in Drosophila eye discs that contain large ci-null mutant clones. Similarly, adult eyes containing large ci mutant clones appear normal both externally and in internal sections. These results, coupled with ectopic expression analysis in the wing disc, suggest that Ciact plays little or no role during normal photoreceptor differentiation. Furthermore, these results support a model in which the major role for Hh signaling during the initiation of photoreceptor differentiation is to prevent the production of Cirep (Pappu, 2003).

Interestingly, ci-null mutant clones that span the furrow do not hasten furrow progression. Although ectopic activation of the Hh pathway is sufficient to induce precocious furrow advancement and photoreceptor differentiation, loss of Ci is not. A likely explanation for this apparent contradiction may be found in the distinction between loss- and gain-of-function experiments. Specifically, although Ciact normally plays little or no role in eye development, ectopic production of Ciact is sufficient to induce precocious furrow advancement. Intriguingly, vertebrate homologs of Drosophila ci have evolved to carry out either activator (Gli1 and Gli2) or repressor (Gli3 and perhaps Gli2) functions independently. These findings demonstrate that in the absence of gene duplication, tissue-specific separation of these functions has also occurred in Drosophila (Pappu, 2003).

It is proposed that Hh signaling acts as a binary switch during Drosophila eye development to control the timing of initiation of photoreceptor differentiation. Specifically, the data suggest that during early larval development Cirep normally inhibits retinal morphogenesis by blocking eya and dpp expression. Hh signaling in late second instar larvae blocks production of Cirep, which in turn allows dpp and eya expression, MF initiation, progression and photoreceptor differentiation. Rather than regulating the differentiation of multiple cell types in a concentration-dependent manner, the data suggest that Hh signaling acts as a molecular switch that is sufficient to initiate dpp and eya expression and retinal morphogenesis. This model also explains the seemingly contradictory phenotypes of loss of smo (blocks MF initiation) and loss of ci (no effect) during Drosophila eye development. Loss of ci creates a permissive environment for eya and dpp expression and photoreceptor differentiation, rendering eye development Hh independent. By contrast, Cirep persists in the absence of smo function and thus photoreceptor morphogenesis does not occur in smo clones. Since ci null mutant clones in the eye develop normally, other Hh independent mechanisms must also act to control the initiation of retinal morphogenesis in Drosophila (Pappu, 2003).

Posterior margin smo mutant clones lack Eya expression and photoreceptor differentiation. The lack of eya expression in these cells is attributed to their inability to block the production of Cirep. Furthermore, the data demonstrate that co-expression of dpp and eya in these posterior smo mutant clones rescues photoreceptor differentiation. In addition, dpp and eya co-expression is sufficient to rescue delayed furrow progression in smo clones. However, the precise temporal and spatial order of photoreceptor recruitment may not be rescued in these clones. Thus, the requirement for Hh signaling in the eye can be circumvented by the expression of the downstream targets dpp and eya. These results demonstrate that eya is a crucial eye-specific target of Hh signaling during the initiation of retinal differentiation and has led to a new model for the initiation of retinal morphogenesis. In this model, Hh signaling blocks the proteolytic degradation of Ciact into Cirep, thus allowing initiation of dpp expression. Once dpp expression is established, the absence of Cirep allows dpp to act in parallel with ey to initiate eya expression, which in turn leads to so expression. Furthermore, dpp cooperates with eya and so to initiate the expression of dac and extensive feedback regulation among these genes leads to consolidation of retinal cell fates (Pappu, 2003).

Low-affinity transcription factor binding sites shape morphogen responses and enhancer evolution

In the era of functional genomics, the role of transcription factor (TF)-DNA binding affinity is of increasing interest: for example, it has recently been proposed that low-affinity genomic binding events, though frequent, are functionally irrelevant. This study investigated the role of binding site affinity in the transcriptional interpretation of Hedgehog (Hh) morphogen gradients. It is noted that enhancers of several Hh-responsive Drosophila genes have low predicted affinity for Ci, the Gli family TF that transduces Hh signalling in the fly. Contrary to an initial hypothesis, improving the affinity of Ci/Gli sites in enhancers of dpp, wingless and stripe, by transplanting optimal sites from the patched gene, did not result in ectopic responses to Hh signalling. Instead, it was found that these enhancers require low-affinity binding sites for normal activation in regions of relatively low signalling. When Ci/Gli sites in these enhancers were altered to improve their binding affinity, patterning defects were observed in the transcriptional response that are consistent with a switch from Ci-mediated activation to Ci-mediated repression. Synthetic transgenic reporters containing isolated Ci/Gli sites confirmed this finding in imaginal discs. It is proposed that the requirement for gene activation by Ci in the regions of low-to-moderate Hh signalling results in evolutionary pressure favouring weak binding sites in enhancers of certain Hh target genes (Ramos, 2013).

This study present in vivo evidence corroborating previous findings that multiple tissue-specific enhancers require low-affinity Ci binding sites for optimal activation by Hh/Ci. Most of the Hh target enhancers identified up to this point in Drosophila and mouse are regulated by degenerate Ci/Gli binding sites of low predicted affinity. The prevalence of these non-consensus sites in Hh target enhancers across species demonstrates their importance in regulating the Hh response. The transcriptional relevance of low-affinity TF binding is not limited to Hh/Ci regulated enhancers. For instance, two phylogenetically conserved low-affinity binding sites in the mouse Pax6 lens enhancer have been shown to be critical to promote gene expression at the right stage of development (Ramos, 2013).

A mechanistic explanation is provided as to why these Hh/Ci-regulated elements require low-affinity sites to activate transcription in cells with moderate signalling levels. Clusters of high-affinity sites mediate a restricted response in cells with high levels of Hh signalling, most likely as a result of cooperative interactions among Ci-Rep molecules in highly occupied Ci binding sites, whereas clusters of low-affinity sites mediate a broader response by having lower occupancy by Ci. Using synthetic enhancer reporters with high- or low-affinity Ci binding sites, this effect was confirmed in the wing, but not in embryos. This tissue-specific discrepancy may imply a context-dependent function for some non-consensus Ci binding sites. As in the Pax6 lens enhancer (Rowan, 2010), it is possible that some low-affinity binding sites are required specifically during earlier stages of development to interpret overall lower levels of Hh signalling (Ramos, 2013).

Finally, clues are provided as to additional regulatory inputs into dppD by showing a requirement for conserved consensus homeodomain (HD) binding sites. Cooperation between Glis and HD proteins has been recently shown in the mouse neural tube. In this case, HD proteins are critical to repress Hh-regulated neural tube enhancers, whereas in dppD they are critical to activate gene expression (Ramos, 2013).

The limited number of known, experimentally confirmed, direct Hh/Gli target enhancers may reflect the widespread, practical tendency to search for consensus or near-consensus motifs, and to focus on the highest peaks of TF-DNA binding, when hunting for cis-regulatory sequences. From a biochemical standpoint—for example, when mining ChIP-seq data—low-affinity DNA-binding interactions are troublesome because they are much more common, by definition, than the top 1% of peaks. It is important to note that iy is not always useful to strictly equate ChIP peak height with TF binding affinity, nor to equate in vitro binding or in silico 'motif quality' with in vivo TF occupancy, though these properties may often be roughly correlated. Separating the weak but functional binding events from weak and non-functional binding events is extremely challenging, and some have proposed that low-affinity genome-binding interactions can be categorically ignored. This certainly simplifies the problem from a computational perspective, but the findings discussed here and elsewhere suggest a risk of discarding functional sequences. Similar challenges confront in silico genomic screens to identify clusters of predicted TF binding sites: these necessarily filter out binding events of low predicted affinity, because there are many more predicted low-affinity binding motifs than consensus high-affinity motifs in any given sequence. Binding site predictions have been supported by taking evolutionary sequence conservation into account, but this risks filtering out true positives: as shown in Ci motif alignments, lower-affinity binding sites seem to be less constrained with respect to sequence variation, even in cases when the presence of the site itself is highly conserved. This is presumably because, for each non-consensus binding motif, there are multiple alternative sequences with similar affinity and thus equivalent functionality. Importantly, this type of degenerate motif conservation is easily missed: for example, some of the well-conserved Ci motifs described in this study are not properly aligned in the UCSC Genome Browser, because they do not constitute contiguous blocks of perfect sequence identity. To avoid these pitfalls, it is important to use phylofootprinting approaches that account for these alignment flaws. In contrast to most of the low-affinity binding sites discussed in this study, optimal-affinity Ci motifs in the ptc enhancer have been preserved throughout the evolution of the genus Drosophila, and perhaps much farther: GACCACCCA motifs occur in promoter-proximal regions of multiple vertebrate orthologues of ptc (Ramos, 2013).

Evolutionary enhancer sequence alignments, along with limited experimental data, also suggest that, although many predicted low-affinity sites are poorly conserved, overall TF occupancy on an enhancer may be maintained despite significant sequence turnover. This may occur either through the rapid gain and loss of individual sites, or through the maintenance of relatively weak binding affinity at a site that is unstable at the level of DNA sequence. While this last idea requires further direct testing, it is consistent with the fact that Gli sites of moderate predicted affinity have many sequence variants of similar quality, whereas the highest-affinity motifs have far fewer alternatives of similar quality. In other words, there are many more ways to be a weak binding site than a strong site. For example, among all possible 9-mer sequences, there are 654 motifs with Ci matrix similarity scores between 70 and 75 (inclusive), but only 12 motifs with scores between 90 and 95, and one motif with a score above 95. Therefore, weaker binding sites, and the enhancers containing them, have a far greater volume of sequence space in which to roam without strongly impacting transcriptional output. A thermodynamics-based simulation of enhancer evolution has shown that there is a greater number of fit solutions using weak TF sites than using high-affinity sites for a given gene expression problem (Ramos, 2013).

Equally consistent with the view of TF binding site evolution is the fact that it is much easier (that is, more likely) to create a low-affinity, non-consensus binding motif with a single mutation than a high-affinity consensus motif. An enhancer-sized DNA sequence can acquire a weak Gli motif with single-nucleotide substitutions at any of a large number of positions, as demonstrated by simulations. These arguments may help to explain why sequence conservation is not a foolproof test of the functional relevance of non-consensus TF binding sites (Ramos, 2013).

While there is no simple answer to the technical challenges facing those who hunt enhancers, the findings described in this report lead to a conclusion that low-affinity TF-DNA interactions, mediated by non-consensus and often poorly conserved sequence motifs, play important and widespread roles in developmental patterning and cis-regulatory evolution, and therefore cannot be safely ignored (Ramos, 2013).

Localized JNK signaling regulates organ size during development

A fundamental question of biology is what determines organ size. Despite demonstrations that factors within organs determine their sizes, intrinsic size control mechanisms remain elusive. This study shows that Drosophila wing size is regulated by JNK signaling during development. JNK is active in a stripe along the center of developing wings, and modulating JNK signaling within this stripe changes organ size. This JNK stripe influences proliferation in a non-canonical, Jun-independent manner by inhibiting the Hippo pathway. Localized JNK activity is established by Hedgehog signaling, where Ci elevates dTRAF1 expression. As the dTRAF1 homolog, TRAF4, is amplified in numerous cancers, these findings provide a new mechanism for how the Hedgehog pathway could contribute to tumorigenesis, and, more importantly, provides a new strategy for cancer therapies. Finally, modulation of JNK signaling centers in developing antennae and legs changes their sizes, suggesting a more generalizable role for JNK signaling in developmental organ size control (Willsey, 2016).

Two independently generated antibodies that recognize the phosphorylated, active form of JNK (pJNK) specifically label a stripe in the pouch of developing wildtype third instar wing discs. Importantly, localized pJNK staining is not detected in hemizygous JNKK mutant discs, in clones of JNKK mutant cells within the stripe, following over-expression of the JNK phosphatase puckered (puc), or following RNAi-mediated knockdown of bsk using two independent, functionally validated RNAi lines (Willsey, 2016).

The stripe of localized pJNK staining appeared to be adjacent to the anterior-posterior (A/P) compartment boundary, a location known to play a key role in organizing wing growth, and a site of active Hedgehog (Hh) signaling. Indeed, pJNK co-localizes with the Hh target gene patched (ptc). Expression of the JNK phosphatase puc in these cells specifically abrogated pJNK staining, as did RNAi-mediated knockdown of bsk. Together, these data indicate that the detected pJNK signal reflects endogenous JNK signaling activity in the ptc domain, a region of great importance to growth control. Indeed, while at earlier developmental stages pJNK staining is detected in all wing pouch cells, the presence of a localized stripe of pJNK correlates with the time when the majority of wing disc growth occurs (1000 cells/disc at mid-L3 stage to 50,000 cells/disc at 20 hr after pupation, so it is hypothesized that localized pJNK plays a role in regulating growth (Willsey, 2016).

Inhibition of JNK signaling in the posterior compartment previously led to the conclusion that JNK does not play a role in wing development. The discovery of an anterior stripe of JNK activity spurred a reexamination of the issue. Since bsk null mutant animals are embryonic lethal, JNK signaling was conditionally inhibited in three independent ways in the developing wing disc. JNK inhibition was achieved by RNAi-mediated knockdown of bsk (bskRNAi#1or2), by expression of JNK phosphatase (puc), or by expression of a dominant negative bsk (bskDN). These lines have been independently validated as JNK inhibitors. Inhibition of JNK in all wing blade cells (rotund-Gal4, rn-Gal4) or specifically in ptc-expressing cells (ptc-Gal4) resulted in smaller adult wings in all cases, up to 40% reduced in the strongest cases. Importantly, expression of a control transgene (UAS-GFP) did not affect wing size. This contribution of JNK signaling to size control is likely an underestimate, as the embryonic lethality of bsk mutations necessitates conditional, hypomorphic analysis. Nevertheless, hypomorphic hepr75/Y animals, while pupal lethal, also have smaller wing discs, as do animals with reduced JNK signaling due to bskDN expression. Importantly, total body size is not affected by inhibiting JNK in the wing. Even for the smallest wings generated (rn-Gal4, UAS-bskDN), total animal body size is not altered (Willsey, 2016).

To test whether elevation of this signal can increase organ size, eiger (egr), a potent JNK activator, was expressed within the ptc domain (ptc-Gal4, UAS-egr). Despite induction of cell death as previously reporte and late larval lethality, a dramatic increase was observed in wing disc size without apparent duplications or changes in the shape of the disc. While changes in organ size could be due to changing developmental time, wing discs with elevated JNK signaling were already larger than controls assayed at the same time point. Similarly, inhibition of JNK did not shorten developmental time. Thus, changes in organ size by modulating JNK activity do not directly result from altering developmental time. Finally, the observed increase in organ size is not due to induction of apoptosis, as expression of the pro-apoptotic gene hid does not increase organ size. In contrast, it causes a decrease in wing size. Furthermore, co-expression of diap1 or p35 did not significantly affect the growth effect of egr expression, while the effect was dependent on Bsk activity (Willsey, 2016).

In stark contrast to known developmental morphogens, no obvious defects were observed in wing venation pattern following JNK inhibition, suggesting that localized pJNK may control growth in a pattern formation-independent manner. Indeed, even a slight reduction in Dpp signaling results in dramatic wing vein patterning defects. Second, inhibiting Dpp signaling causes a reduction in wing size along the A-P axis, while JNK inhibition causes a global reduction. Furthermore, ectopic Dpp expression increases growth in the form of duplicated structures, while increased JNK signaling results in a global increase in size. Molecularly, it was confirmed that reducing Dpp signaling abolishes pSMAD staining, while quantitative data shows that inhibiting JNK signaling does not. Furthermore, it was also found that Dpp is not upstream of pJNK, as reduction in Dpp signaling does not affect pJNK. Together, the molecular data are consistent with the phenotypic results indicating that pJNK and Dpp are separate programs in regulating growth. Consistent with these findings it has been suggested that Dpp does not play a primary role in later larval wing growth control (Akiyama, 2015). Finally, it was found that inhibition of JNK does not affect EGFR signaling (pERK) and that inhibition of EGFR does not affect the establishment of pJNK (Willsey, 2016).

A difference in size could be due to changes in cell size and/or number. Wings with reduced size due to JNK inhibition were examined and no changes in cell size or apoptosis were found, suggesting that pJNK controls organ size by regulating cell number. Consistently, the cell death inhibitor p35 was unable to rescue the decreased size following JNK inhibition. Indeed, inhibition of JNK signaling resulted in a decrease in proliferation, while elevation of JNK signaling in the ptc domain resulted in an increase in cell proliferation in the enlarged wing disc. Importantly, this increased proliferation is not restricted to the ptc domain, consistent with previous reports that JNK can promote proliferation non-autonomously (Willsey, 2016).

To determine the mechanism by which pJNK controls organ size, canonical JNK signaling through its target Jun was considered. Interestingly, RNAi-mediated knockdown of jun in ptc cells does not change wing size, consistent with previous analysis of jun mutant clones in the wing disc. Furthermore, in agreement with this, a reporter of canonical JNK signaling downstream of jun (puc-lacZ) is not expressed in the pJNK stripe. Finally, knockdown of fos (kayak, kay) alone or with junRNAi did not affect wing size. Together, these data indicate that canonical JNK signaling through jun does not function in the pJNK stripe to regulate wing size (Willsey, 2016).

In search of such a non-canonical mechanism of JNK-mediated size control, the Hippo pathway was considered. JNK signaling regulates the Hippo pathway to induce autonomous and non-autonomous proliferation during tumorigenesis and regeneration via activation of the transcriptional regulator Yorkie (Yki). Recently it has been shown that JNK activates Yki via direct phosphorylation of Jub. To test whether this link between JNK and Jub could account for the role of localized pJNK in organ size control during development, RNAi-mediated knockdown of jub was performed in the ptc stripe, and adults with smaller wings were observed. Indeed, the effect of JNK loss on wing size can be partially suppressed in a heterozygous lats mutant background and increasing downstream yki expression in all wing cells or just within the ptc domain can rescue wing size following JNK inhibition. These results suggest that pJNK controls Yki activity autonomously within the ptc stripe, leading to a global change in cell proliferation. This hypothesis predicts that the Yki activity level within the ptc stripe influences overall wing size. Consistently, inhibition of JNK in the ptc stripe translates to homogeneous changes in anterior and posterior wing growth. Similarly, overexpression or inhibition of Yki signaling in the ptc stripe also results in a global change in wing size (Willsey, 2016).

It is important to note that the yki expression line used is wild-type Yki, which is still affected by JNK signaling. For this reason, the epistasis experiment was also performed with activated Yki, which is independent of JNK signaling. Expression of this activated Yki in the ptc stripe caused very large tumors and lethality. Importantly, inhibiting JNK in this context did not affect the formation of these tumors or the lethality. Furthermore, inhibiting both JNK and Yki together does not enhance the phenotype of Yki inhibition alone, further supporting the idea that Yki is epistatic to JNK, instead of acting in parallel processes (Willsey, 2016).

Mutants of the Yki downstream target four-jointed (fj) have small wings with normal patterning, and fj is known to propagate Hippo signaling and affect proliferation non-autonomously. Although RNAi-mediated knockdown of fj in ptc cells does not cause an obvious change in wing size, it is sufficient to block the Yki-induced effect on increasing wing size . However, overexpression of fj also reduces wing size, which makes it not possible to test for a simple epistatic relationship. Overall, these data are consistent with the notion that localized pJNK regulates wing size not by Jun-dependent canonical JNK signaling, but rather by Jun-independent non-canonical JNK signaling involving the Hippo pathway (Willsey, 2016).

While morphogens direct both patterning and growth of developing organs, a link between patterning molecules and growth control pathways has not been established. pJNK staining is coincident with ptc expression, suggesting it could be established by Hh signaling. During development, posterior Hh protein travels across the A/P boundary, leading to activation of the transcription factor Cubitus interruptus (Ci) in the stripe of anterior cells. To test whether localized activation of JNK is a consequence of Hh signaling through Ci, RNAi-mediated knockdown of ci was performed, and it was found that the pJNK stripe is eliminated. Consistently, adult wing size is globally reduced. In contrast, no change was observed in pJNK stripe staining following RNAi-mediated knockdown of dpp or EGFR. Expression of non-processable Ci leads to increased Hh signaling. Expression of this active Ci in ptc cells leads to an increase in pJNK signal and larger, well-patterned adult wings. The modest size increase shown for ptc>CiACT is likely due to the fact that higher expression of this transgene (at 25 ° C) leads to such large wings that pupae cannot emerge from their cases. For measuring wing size, this experiment was performed at a lower temperature so that the animals were still viable. Furthermore, inhibition of JNK in wings expressing active Ci blocks Ci's effects, and resulting wings are similar in size to JNK inhibition alone . Together, these data indicate that Hh signaling through Ci is responsible for establishing the pJNK stripe (Willsey, 2016).

To determine the mechanism by which Ci activates the JNK pathway, transcriptional profiles of posterior and ptc domain cells isolated by FACS from third instar wing discs were compared. Of the total 12,676 unique genes represented on the microarray, 50.4% (6,397) are expressed in ptc domain cells, posterior cells, or both. Hh pathway genes known to be differentially expressed were identified. It was next asked whether any JNK pathway genes are differentially expressed, and and it was found that dTRAF1 expression is more than five-fold increased in ptc cells, while other JNK pathway members are not differentially expressed (Willsey, 2016).

dTRAF1 is expressed along the A/P boundary and ectopic expression of dTRAF1 activates JNK signaling. Thus, positive regulation of dTRAF1 expression by Ci could establish a stripe of pJNK that regulates wing size. Indeed, Ci binding motifs were identified in the dTRAF1 gene, and a previous large-scale ChIP study confirms a Ci binding site within the dTRAF1 gene. Consistently, a reduction in Ci led to a 29% reduction in dTRAF1 expression in wing discs. Given that the reduction of dTRAF1 expression in the ptc stripe is buffered by Hh-independent dTRAF1 expression elsewhere in the disc, this 29% reduction is significant. Furthermore, inhibition of dTRAF1 by RNAi knockdown abolished pJNK staining. Finally, these animals have smaller wings without obvious pattern defects. Conversely, overexpression of dTRAF1 causes embryonic lethality, making it not possible to attempt to rescue a dTRAF1 overexpression wing phenotype by knockdown of bsk. Nevertheless, it has been shown that dTRAF1 function in the eye is Bsk-dependent. Finally, inhibition of dTRAF1 modulates the phenotype of activated Ci signaling. Together, these data reveal that the pJNK stripe in the developing wing is established by Hh signaling through Ci-mediated induction of dTRAF1 expression (Willsey, 2016).

Finally, localized centers of pJNK activity were detected during the development of other imaginal discs including the eye/antenna and leg. Inhibition of localized JNK signaling during development caused a decrease in adult antenna size and leg size. Conversely, increasing JNK signaling during development resulted in pupal lethality; nevertheless, overall sizes of antenna and leg discs were increased. Together, these data indicate that localized JNK signaling regulates size in other organs in addition to the wing, suggesting a more universal effect of JNK on size control (Willsey, 2016).

Intrinsic mechanisms of organ size control have long been proposed and sought after. This study reveals that in developing Drosophila tissues, localized, organ-specific centers of JNK signaling contribute to organ size in an activity level-dependent manner. Such a size control mechanism is qualitatively distinct from developmental morphogen mechanisms, which affect both patterning and growth. Aptly, this mechanism is still integrated in the overall framework of developmental regulation, as it is established in the wing by the Hh pathway. These data indicate that localized JNK signaling is activated by Ci-mediated induction of dTRAF1 expression. Furthermore,it is not canonical Jun-dependent JNK signaling, but rather non-canonical JNK signaling that regulates size, possibly through Jub-dependent regulation of Yki signaling, as described for regeneration. As the human dTRAF1 homolog, TRAF4, and Hippo components are amplified in numerous cancers, these findings provide a new mechanism for how the Hh pathway could contribute to tumorigenesis. More importantly, these findings offer a new strategy for potential cancer therapies, as reactivating Jun in Hh-driven tumors could lead tumor cells towards an apoptotic fate (Willsey, 2016).

cubitus interruptus: Biological Overview | Evolutionary Homologs | Regulation | Protein Interactions | Developmental Biology | Effects of Mutation | References

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