cis-Regulatory Sequences and Functions

A functional approach has been taken to define the minimal regions of both the Cubitus interruptus protein and the cis-acting regulatory regions of the gene patched, sufficient to mediate its transcriptional activation. The zinc finger domain of CI alone, when fused to the herpes simplex activation domain, can activate transcription of patched in imaginal discs, indicating that the specificity of CI activity is determined by its putative DNA binding domain. In addition, CI can serve as a transcriptional activator in a yeast synthetic promoter. Finally, a 758-bp patched upstream regulatory element, that directs robust expression along the anteroposterior compartment boundary, contains three consensus CI zinc finger binding sites, which when mutated completely abolish expression. All told, circumstantial evidence is leaning to the conclusion that CI acts directly as a transcription factor to regulate Hedgehog target genes (Alexandre, 1996).

The cis-regulatory logic of Hedgehog gradient responses: key roles for gli binding affinity, competition, and cooperativity

Gradients of diffusible signaling proteins control precise spatial patterns of gene expression in the developing embryo. This study used quantitative expression measurements and thermodynamic modeling to uncover the cis-regulatory logic underlying spatially restricted gene expression in a Hedgehog (Hh) gradient in Drosophila. When Hh signaling is low, the Hh effector Gli, known as Cubitus interruptus (Ci) in Drosophila, acts as a transcriptional repressor; when Hh signaling is high, Gli acts as a transcriptional activator. Counterintuitively and in contrast to previous models of Gli-regulated gene expression, this study found that low-affinity binding sites for Ci were required for proper spatial expression of the Hh target gene decapentaplegic (dpp) in regions of low Hh signal. Three low-affinity Ci sites enabled expression of dpp in response to low signal; increasing the affinity of these sites restricted dpp expression to regions of maximal signaling. A model incorporating cooperative repression by Ci correctly predicted the in vivo expression of a reporter gene controlled by a single Ci site. This work clarifies how transcriptional activators and repressors, competing for common binding sites, can transmit positional information to the genome. It also provides an explanation for the widespread presence of conserved, nonconsensus Gli binding sites in Hh target genes (Parker, 2011).

The enhancers of dpp and ptc exhibit a regulatory logic opposite that predicted by the activator threshold model. ptc is regulated by Ci sites that match the optimal binding sequence (GACCACCCA), whereas dpp is regulated by nonconsensus sites of low predicted affinity. Competitive electrophoretic mobility shift assays (EMSAs) were used to measure the relative in vitro affinities of Ci sites in the ptc and dpp enhancers, and it was found that Ciptc sites in the ptc enhancer have considerably higher affinity than Cidpp sites. The predicted superior affinity of Ciptc sites, relative to Cidpp sites, is conserved across 12 Drosophila species. Thus, the regulation of dpp and ptc in the wing is opposite to that predicted by a simple activator threshold model. ptc, which is restricted to the region of highest Hh signal, is regulated by high-affinity sites. In contrast, dpp, which responds more broadly in a zone of lower Hh signaling, is regulated by low-affinity sites (Parker, 2011).

To investigate the developmental role of the low-affinity sites in the dpp enhancer, all three sites were altered to match the high-affinity Ci binding sequence found in the ptc enhancer, a change of only seven nucleotides. Transgenic lines were created containing an extra Flp-inducible copy of dpp, driven by the dpp disc (dppD) enhancer containing either wild-type low-affinity (Ciwt) or altered high-affinity (Ciptc) sites. An extra copy of dpp driven by the low-affinity dppD-Ciwt enhancer had no effect on development or survival, whereas the high-affinity Ciptc enhancer caused lethal developmental defects that resemble the effects of dpp misexpression in imaginal discs, including severe head and limb deformities and pupal lethality resulting from overgrowth fusion, and patterning defects in antenna and leg discs. These results indicate that the conserved low affinity of the dppD enhancer for Ci is functionally relevant (Parker, 2011).

A quantitative reporter gene assay was developed to further explore the role of low-affinity Ci binding sites in the dpp wing disc enhancer. Transgenic fly lines were constructed carrying two reporter genes: dppD-Ciptc-RFP, consisting of the high-affinity version of the dpp enhancer driving expression of a red fluorescent protein (RFP), and one of several dpp enhancers driving green fluorescent protein (GFP). By measuring GFP fluorescence across a transect of the wing pouch and normalizing to peak RFP expression in each disc, a quantitative readout was obtained of both the position and the intensity of GFP reporter activity (Parker, 2011).

Using this assay, the activity was compared of different versions of the dppD enhancer driving GFP expression, containing either three low-affinity sites (dppD-Ciwt) or three high-affinity sites (dppD-Ciptc). Ci-independent, 'basal' expression was also measured from a construct (dppD-CiKO) in which all three Ci sites were mutated to abolish Ci binding. This basal expression captures the effects of all factors other than Ci on dpp, including Engrailed, which directly represses dpp near the anterior/posterior boundary. This basal construct enabled direct measurement of both activation and repression by Ci, by comparing the activity of the low- or high-affinity enhancers against that of dppD-CiKO. The results show that the response of dpp to Hh cannot be explained by an activator threshold model. High-affinity Ciptc sites caused a posterior shift in stripe position toward the region of strongest Hh signal, whereas low-affinity Ciwt sites produced stronger activation in regions of moderate Hh signal. When the basal dppD-CiKO-GFP expression was subtracted from that of dppD-Ciptc-GFP and dppD-Ciwt-GFP, it was observed that within the zone of moderate Hh signal, low-affinity sites produced activation, whereas high-affinity sites conferred repression. This observation shows that CiREP plays a substantial role in the response to moderate Hh signal, a finding that directly contradicts the assumptions of the activator threshold model. Thus, an alternate biophysical model is required to explain the regulatory logic of the dpp response to Hh (Parker, 2011).

It is concluded that spatial information in the wing disc Hh gradient is interpreted by a cis-regulatory logic that relies on activator-repressor competition, which is modulated by binding site affinity and cooperative repression. In previous studies of Hh target genes, the role of the affinity of the Gli or Ci binding site has been neglected or has been assumed to play a role opposite to what the current data show. Moreover, the currently accepted activator threshold model of the transcriptional response to Hh assumes that the role of Gli and Ci repressors is limited to regions of little or no Hh signal. No previously described model of Hh response, including the activator threshold model, can account for the observations described in this study. The data show that substantial repression can occur even at moderate Hh signal and suggest that the transcriptional response in much of the Hh gradient depends on the outcome of a competition between CiACT and CiREP for enhancer binding. This new model of the cis-regulatory logic underlying Hh response integrates the effects of both CiACT and CiREP along the entire Hh gradient and explains the importance of low-affinity Gli binding sites in the positioning of gene expression (Parker, 2011).

The results suggest that the low affinity of the dpp enhancer for Ci can be explained by the need to mitigate the effects of cooperative repression in a region of the gradient where substantial amounts of CiREP are present, while still allowing activation by CiACT. Within the context of this model, repressor cooperativity is defined as any interaction that makes the binding of additional CiREP more favorable when one CiREP is already bound. Cooperativity could arise from direct interactions between CiREP or from interactions of CiREP with other transcription factors, cofactors, or histones. In principle, CiREP cooperativity at the enhancer could be attenuated by various cis-regulatory strategies besides lowering binding affinity, such as reducing the number of Ci sites or increasing their spacing. However, these alternative strategies may not be equally able to maintain activation by CiACT in regions of low Hh signaling (Parker, 2011).

The posterior-to-anterior gradient of Hh in the wing disc establishes opposing gradients of CiACT and CiREP. High amounts of CiACT are present at the anterior/posterior boundary, whereas more anterior regions feature high amounts of CiREP. Within the intermediate zone of the gradient, mixed amounts of CiACT and CiREP compete for enhancer binding, with activation and repression determined by the ratio of bound CiACT to bound CiREP. In the repressor cooperativity model, repressors outcompete activators for binding at high-affinity enhancers, but not at low-affinity enhancers, in this region of the gradient. Several morphogen signaling pathways have the potential to produce reciprocal gradients of repressors and activators competing for common binding sites. This study has presented the first detailed mechanistic model that explains how reciprocal gradients of Gli activators and repressors are transcriptionally interpreted. A similar regulatory logic may inform responses to other morphogens that control transcriptional switches, particularly those whose target genes are regulated by low-affinity sites (Parker, 2011).

The model provides an explanation for the widespread presence of evolutionarily conserved, nonconsensus Gli or Ci binding sites in the enhancers of Hh target genes. With the exception of ptc, all known direct targets of Hh in Drosophila are regulated by nonconsensus Ci sites with predicted low affinity The results indicate that low-affinity sites are necessary to position a stripe of expression in the middle of a Hh gradient, because high-affinity sites induce repression outside the zone of strongest signaling. Because mammalian Gli target genes are also regulated by nonconsensus sites, the conclusions may also apply to vertebrate Hh targets: Such targets may acquire weak-affinity Gli sites to minimize cooperative repression in a Gli cross-gradient (Parker, 2011).

In a few documented cases, low-affinity transcription factor binding sites have important spatiotemporal patterning functions. Two well-studied examples are the response to the morphogen Dorsal in Drosophila and the temporal control of developmental gene expression. In these cases, low-affinity sites set a high threshold for activator concentration, restricting activation to cells or times in which activator concentration is maximal. In the case described in this study, an opposite cis-regulatory logic applies: Low-affinity sites are specifically required for activation in cells receiving lower amounts of signal. This is a consequence of the fact that in this region of the gradient, activators and repressors compete for the same genomic binding sites (Parker, 2011).

The results suggest that most current biochemical and computational approaches to identifying Hh target genes, which typically focus on the highest-affinity Ci or Gli sites, may overlook a large proportion of important Hh target genes. More generally, transcriptional cooperativity may play an important cis-regulatory role in enhancers with conserved low-affinity binding sites (Parker, 2011).

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).

Kto-Skd complex can regulate ptc expression by interacting with Ci in Hedgehog signaling pathway

Hedgehog (Hh) signaling pathway plays a very important role in metazoan development by controlling pattern formation. Drosophila imaginal discs are subdivided into anterior and posterior compartments which derive from adjacent cell populations. The anterior/posterior (A/P) boundaries which are critical to maintain the position of organizers are established by a complex mechanism involving the Hh signaling. This study uncovered the regulation of ptc in Hh signaling pathway which is contributive to A/P boundary formation by two subunits of mediator complex, Kto and Skd, which can also regulate boundary location. Collectively, further evidence is provided that Kto-Skd affects the A/P-axial development of the whole wing disc. And Kto can interact with Cubitus interruptus (Ci) and bind to the Ci-binding region on the ptc promoter. Both subunits are regulated by Hh signals to downregulate ptc expression (Mao, 2014).

Transcriptional Regulation

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

The effects of ectopic Cubitus interruptus 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 anteior 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).

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)

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

Reduced Protein kinase A (PKA) activity in anterior imaginal disc cells leads to cell-autonomous induction of decapentaplegic, wingless, and patched transcription that is independent of hedgehog gene activity. Expression of a mutant regulatory subunit to anterior cells at the AP border of the wing imaginal disc results in localized PKA inactivation and can substitute for hh in promoting disc growth. PKA inhibition in anterior cells of the AP border can induce patched expression and can thus substitute for the growth-promoting activity of hh during larval life (Li, 1995).

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

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

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

The two signaling proteins, Wingless and Hedgehog, play fundamental roles in patterning cells within each metamere of the Drosophila embryo. Within the ventral ectoderm, Hedgehog signals both to the anterior and posterior directions: anterior flanking cells express the wingless and patched Hedgehog target genes whereas posterior flanking cells express only patched. Furthermore, Hedgehog acts as a morphogen to pattern the dorsal cuticle, on the posterior side of cells where it is produced. Thus responsive embryonic cells appear to react according to their position relative to the Hedgehog source. The molecular basis of these differences is still largely unknown. In this paper it is shown that one component of the Hedgehog pathway, the kinase Fused accumulates preferentially in cells that could respond to Hedgehog but that Fused concentration is not a limiting step in the Hedgehog signaling. Direct evidence is presented that Fused is required autonomously in anterior cells neighboring Hedgehog in order to maintain patched and wingless expression, while in turn, Wingless is maintaining engrailed and hedgehog expression. By expressing different components of the Hedgehog pathway only in anterior, wingless-expressing cells, it could shown that the Hedgehog signaling components Smoothened and Cubitus interruptus are required in cells posterior to Hedgehog domain to maintain patched expression, whereas Fused is not necessary in these cells. This result suggests that Hedgehog responsive ventral cells in embryos can be divided into two distinct types depending on their requirement for Fused activity. In addition, the morphogen Hedgehog can pattern the dorsal cuticle independent of Fused. In order to account for these differences in Fused requirements, the existence of position-specific modulators of the Hedgehog response is proposed (Thérond, 1999).

The manner in which Hh molecules regulate a target cell remains poorly understood. In the Drosophila embryo, Hh is produced in identical stripes of cells in the posterior compartment of each segment. From these cells a Hh signal acts in both anterior and posterior directions. In the anterior cells, the target genes wingless and patched are activated whereas posterior cells respond to Hh by expressing rhomboid and patched. This study examines the role of the transcription factor Cubitus interruptus (Ci) in this process. So far, Ci has been thought to be the most downstream component of the Hh pathway, capable of activating all Hh functions. However, the study of a null ci allele indicates that it is actually not required for all Hh functions. Whereas Hh and Ci are both required for patched expression, the target genes wingless and rhomboid have unequal requirements for Hh and Ci activity. Hh is required for the maintenance of wingless expression before embryonic stage 11 whereas Ci is necessary only later during stage 11. For rhomboid expression Hh is required positively whereas Ci exhibits negative input. These results indicate that factors other than Ci are necessary for Hh target gene regulation. Evidence is presented that the zinc-finger protein Teashirt is one candidate for this activity. It is required positively for rhomboid expression and Teashirt and Ci act in a partially redundant manner before stage 11 to maintain wingless expression in the trunk (Gallet, 2000).

In conclusion, Hh requires at least two different transcription factors during Drosophila embryogenesis to regulate its multiple target genes and to instruct cells with precise behaviors. The transcription factors may act independently (e.g. Ci for ptc; Tsh for rho), cooperatively (e.g. Ci and Tsh for wg maintenance during the cell specification phase) or redundantly (e.g. Ci and Tsh for wg maintenance earlier during the stabilization phase). The possibility that other transcription factors like gooseberry might be recruited for Hh signaling cannot be excluded, especially since denticle density is weaker in tsh;ci double mutants as compared with hh single mutants. Furthermore the dorsal phenotypes of the tsh;ci double mutants are weaker than those of hh. (1) wg transcripts are still present in dorsal patches in tsh;ci mutations whereas they are not present in hh embryos. (2) Dorsal cuticle is not as severely perturbed in tsh;ci larvae as compared with hh null ones (Gallet, 2000).

Finally, pathway bifurcations are involved not only at the level of the transcription factors. The Fu kinase, which is normally required to transduce Hh signal and to convert Ci 155 into a putative Ci act form, is not necessary in all Hh-receiving cells during embryogenesis. While Fu is involved anteriorly to En/Hh-expressing cells for the maintenance of wg and ptc, it is not involved posteriorly for the maintenance of ptc. These results correlate with the Ci isoforms detected: anteriorly the putative Ci act form is present but posteriorly only the Ci 155 form is detected (Gallet, 2000 and references therein).

Engrailed is a nuclear regulatory protein with essential roles in embryonic segmentation and wing morphogenesis. One of its regulatory targets in embryos has been shown to be the Polycomb group gene, polyhomeotic. Transheterozygous adult flies, mutant for both engrailed and polyhomeotic, show a gap in the fourth vein. In the corresponding larval imaginal discs, a polyhomeotic-lacZ enhancer trap is not normally activated in anterior cells adjacent to the anterior-posterior boundary. This intermediary region corresponds to the domain of low engrailed expression that appears in the anterior compartment, during L3. This en expression depends on the putative serine-threonine kinase protein fused and on the level of hh expression in the posterior cells abutting the A/P boundary, and so depends indirectly on en expression in the posterior compartment. The exact role of this late L3 anterior compartment en expression is still not understood (Maschat, 1998).

Several arguments show that engrailed is responsible for the induction of polyhomeotic in these cells. The role of polyhomeotic in this intermediary region is apparently to maintain the repression of hedgehog in the anterior cells abutting the anterior-posterior boundary, since these cells ectopically express hedgehog when polyhomeotic is not activated. Analysis of the expression patterns of different genes of the Hh signaling pathway that are normally expressed in this intermediary region showed that the segmentation gene patched is highly affected in ph/en mutant discs. The gap in the fourth vein can therefore be correlated with a misregulation of ptc in the posterior compartment. Interestingly, this ectopic ptc expression appears not only in the cells where ph is affected, but also in neighboring posterior cells. This ectopic expression of ptc progressively invades the posterior compartment during the third instar to fill the whole compartment in mature larvae. Genetic data indicate that the level of hh expression is involved in this phenomenon, suggesting that the progressive invasion of the posterior compartment by Ptc is due to an increased secretion of Hh by the cells of the anterior intermediary region, towards cells localized more posteriorly. As a consequence of this ptc misregulation, cubitus-interruptus (ci) and decapentaplegic (dpp) are activated in the posterior compartment, suggesting that the intermediary region, where dpp expression is normally confined, expands posteriorly. As a result of the absence of ph activation by En in cells abutting the A/P boundary, this boundary is not maintained at its normal position, but is progressively shifted posteriorly, while cells lose their posterior identity. Thus posterior cells express a new set of genes that are normally characteristic of anterior cells, suggesting a change in the cell identity. Altogether, these data indicate that engrailed and polyhomeotic interactions are required to maintain the anterior-posterior boundary and the posterior cell fate, just prior to the evagination of the wing (Maschat, 1998).

Considering that hh is responsible for the changes appearing in the posterior compartment of ph/en flies implies that posterior cells might become competent to respond to the Hh signal. Such competence could be attributed to the presence of a low level of posterior compartment ci, which is present ectopically in a [ph-; en-/+] background. Indeed, ph has been shown to be a repressor of ci in the posterior compartment and now it seems both en and ph are likely to be responsible for ci repression in the posterior compartment. Transcriptional repression of ci in the posterior compartment could be initiated by en and maintained by ph, the ph expression depending itself on en expression. Indeed, posterior heterozygous en/+ cells do not show any phenotype unless they are also mutant for ph. One could hypothesize a feedback loop involving en and ph to maintain the level of en expression and ci repression in the posterior compartment. If the basic level of en expression in the posterior compartment depends on both en and ph, en could be maintained at a lower level in a [ph-; en-/+] background. These cells might now produce enough Ci and Ptc to become competent to receive the Hh signal. If posterior cells are not competent to receive an Hh signal, higher amounts of Hh would not affect the posterior cells. Such a feedback loop mechanism between en and ph, maintaining the level of en expression, could also explain the lack of hypomorphic en mutants, since such mutants would be detectable only when ph is affected (Maschat, 1998).

The cell surface receptor Notch is required during Drosophila embryogenesis for production of epidermal precursor cells. The secreted factor Wingless is required for specifying different types of cells during differentiation of tissues from these epidermal precursor cells. The results reported here show that the full-length Notch and a form of Notch truncated in the amino terminus associate with Wingless in S2 cells and in embryos. In S2 cells, Wingless and the two different forms of Notch regulate expression of Frizzled 2, a receptor of Wg; hairy, a negative regulator of achaete expression; shaggy, a negative regulator of engrailed expression, and patched, a negative regulator of wingless expression. Analyses of expression of the same genes in mutant N embryos indicate that the pattern of gene regulations observed in vitro reflects regulations in vivo. These results suggest that the strong genetic interactions observed between Notch and wingless genes during Drosophila development is at least partly due to regulation of expression of cuticle patterning genes by Wingless and the two forms of Notch (Wesley, 1999).

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).

ptc is regulated by Hh exclusively via Ci[act] and 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).

Notch and Wingless modulate the response of cells to Hedgehog signaling in the Drosophila wing

During Drosophila wing development, Hedgehog (Hh) signaling is required to pattern the imaginal disc epithelium along the anterior-posterior (AP) axis. The Notch (N) and Wingless (Wg) signaling pathways organise the dorsal-ventral (DV) axis, including patterning along the presumptive wing margin. A functional hierarchy of these signaling pathways is described that highlights the importance of the competing influences of Hh, N, and Wg in establishing gene expression domains. Investigation of the modulation of Hh target gene expression along the DV axis of the wing disc has revealed that collier/knot (col/kn), patched, and decapentaplegic are repressed at the DV boundary by N signaling. Attenuation of Hh signaling activity caused by loss of fused function results in a striking down-regulation of col, ptc, and engrailed (en) symmetrically about the DV boundary. This down-regulation depends on activity of the canonical Wg signaling pathway. It is proposed that modulation of the response of cells to Hh along the future proximodistal (PD) axis is necessary for generation of the correctly patterned three-dimensional adult wing. These findings suggest a paradigm of repression of the Hh response by N and/or Wnt signaling that may be applicable to signal integration in vertebrate appendages (Glise, 2002).

Short-range Hh signaling, partly through activation of Col function, is essential for correct AP patterning and differentiation of L3-L4 intervein tissue. N and Wg first define the DV boundary and later subdivide the region near this boundary into a number of distinct subregions that will eventually differentiate into wing margin bristles and vein tissue. These signals overlap spatially and temporally and lead to opposite fates. It is proposed that in and close to the DV boundary, N, Wg, and Hh signaling exist in a delicate balance to allow vein tissue, bristle, and sensory organ differentiation along the adult wing margin (Glise, 2002).

The Hh target genes col/kn and ptc, in contrast to en, are repressed in a wild type wing in cells corresponding to the presumptive wing margin. It has been demonstrated, using both gain- and loss-of-function experiments, that this repression is mediated by N signaling and that its inhibition results in aberrant morphogenesis of the wing. Hh signaling, achieved either by overexpression of Hh or loss of Ptc activity, is not sufficient to give maximum activation of Hh targets in cells of the prospective wing margin, suggesting that a finely tuned balance of activation and repression is required to achieve the appropriate biological output. However, overexpression of a stabilized form of Ci under the ptc-Gal4 driver results in the activation of Col in the prospective wing margin and defects in wing margin differentiation, indicating that N repression can be overcome by hyperactivity of the Hh signaling pathway. N signaling may lead to the repression of col, ptc, and dpp directly or it may act indirectly by affecting the ability of Ci to act as a transcriptional activator. Since expression of en, which requires the highest level of Hh signaling and Ci activity, appears immune to N repression, the former possibility is favored (Glise, 2002).

Temporal modulation of the Hedgehog morphogen gradient by a patched-dependent targeting to lysosomal compartment

Hedgehog family members are secreted proteins involved in numerous patterning mechanisms. Different posttranslational modifications have been shown to modulate Hedgehog biological activity. The role of these modifications in regulating subcellular localization of Hedgehog has been investigated in the Drosophila embryonic epithelium. Cholesterol modification of Hedgehog is responsible for Hedgehog assembly in large punctate structures and apical sorting through the activity of the sterol-sensing domain-containing Dispatched protein. Movement of these specialized structures through the cellular field is contingent upon the activity of proteoglycans synthesized by the heparan sulfate polymerase Tout-Velu. Finally, the Hedgehog large punctate structures are necessary only for a subset of Hedgehog target genes across the parasegmental boundary, suggesting that presentation of Hedgehog from different membrane compartments is responsible for Hedgehog's functional diversity in epithelial cells (Gallet, 2003).

The repeated pattern of the Drosophila larval ectoderm (which secretes cuticle) has been used to follow Hh activity. Each abdominal segment is composed of two types of cuticle: the naked (or smooth) cuticle and the denticle belts, subdivided into six rows of denticles, easily identifiable by their orientation and shape. This cuticle pattern is under the control of several signaling pathways that are indirectly regulated by Hh. Engrailed (En) controls hh expression in the two rows of cells that define the posterior compartment of the segment. Across the parasegmental boundary (in cells anterior to the En/Hh domain), Hh maintains wingless (wg) transcription in one row of cells. The Wg signal then controls the specification of the naked cuticle. Posterior to the En/Hh domain, Hh initiates rhomboid (rho) transcription in one to two rows of cells. rho activation induces EGF signaling, allowing differentiation of denticles 1-4. Finally, Hh and Wg are required for serrate (ser) repression and restrict its expression in three rows of cells posterior to the rho-expressing cells. Ser initiates a third row of rho expression in adjacent cells. The Hh receptor Patched (Ptc) is also transcriptionally upregulated by the Hh pathway in cells on both sides of the En/Hh domain (Gallet, 2003).

Loss of hh results in loss of both naked cuticle and denticle diversity. This cuticle phenotype correlates with Hh target gene expression: loss of wg, extension of the ser expression domain (which now covers most of the segment) and absence of ptc upregulation. rho expression is strongly reduced, though some remains under the control of Ser. Conversely, ubiquitous expression of full-length hh (HhFL) in the ectoderm with the GAL4-UAS system induces an expansion over four to five cells of both wg and rho expression in the anterior and posterior directions, respectively, while ser expression is completely repressed. Accordingly, the denticle belts of these embryos contain several rows of type 2 denticles, reflecting a uniform level of rho expression in response to a uniform level of Hh. Thus, wg, rho, ser, and ptc expressions reflect direct Hh activity in cells anterior and posterior to en/hh-expressing cells (Gallet, 2003).

Two endogenous Hh isoforms are present in vivo: one bearing both posttranslational lipid modifications and another modified only by a cholesterol adduct. To address the role of these different modifications in Hh signaling, the biological activity of different Hh constructs that do not undergo all modifications was assessed (Gallet, 2003).

It is hypothesized that the differences observed could be accounted for by differential activation mechanisms. These results outline the important role of the Hh cholesterol modification in stimulating the anterior target genes wg and ptc across the parasegmental boundary and, subsequently, naked cuticle differentiation, while cholesterol appears dispensable for posterior induction of ptc and rho and, thus, denticle diversity. Because some wg expression can still be activated by Hh-N, the presence of cholesterol modification on Hh might not be the only requirement for anterior target gene regulation. Hh-N-CD2 and Hh-N-GPI activities suggest that the differences observed could be a consequence of Hh differential sorting in the producing cells and/or access and presentation to the target cell surface (Gallet, 2003).

Differential activation of wg and ptc in anterior cells and of rho and ptc in posterior cells is related to the membrane localization of Hh. Cholesterol-dependent LPS formation and apical targeting are shown to be necessary for proper anterior wg activation but dispensable for rho expression in posterior cells. Conversely, basolateral targeting of Hh in cells producing Hh-N-CD2 and Hh-N-GPI is sufficient to activate the posterior rho expression, independent of the presence of cholesterol (Gallet, 2003).

Differential regulation of Hedgehog target gene transcription by Costal2 and Suppressor of Fused

The mechanism by which the secreted signaling molecule Hedgehog (Hh) elicits concentration-dependent transcriptional responses from cells is not well understood. In the Drosophila wing imaginal disc, Hh signaling differentially regulates the transcription of target genes decapentaplegic (dpp), patched (ptc) and engrailed (en) in a dose-responsive manner. Two key components of the Hh signal transduction machinery are the kinesin-related protein Costal2 (Cos2) and the nuclear protein trafficking regulator Suppressor of Fused [Su(fu)]. Both proteins regulate the activity of the transcription factor Cubitus interruptus (Ci) in response to the Hh signal. This study analyzed the activities of mutant forms of Cos2 in vivo and found effects on differential target gene transcription. A point mutation in the motor domain of Cos2 results in a dominant-negative form of the protein that derepresses dpp but not ptc. Repression of ptc in the presence of the dominant-negative form of Cos2 requires Su(fu), which is phosphorylated in response to Hh in vivo. Overexpression of wild-type or dominant-negative cos2 represses en. These results indicate that differential Hh target gene regulation can be accomplished by differential sensitivity of Cos2 and Su(Fu) to Hh (Ho, 2005).

The data suggest that the activities of Cos2 and Su(fu) are independently regulated by different concentrations of Hh along the gradient that forms from posterior to anterior. In the anterior cells distant from the AP boundary, little or no Hh is received and target genes are silent. In these cells, Cos2 is required for proteolytic processing of Ci into its repressor form and possibly for the delivery of CiFL for lysosomal degradation. The data suggest that Cos2 requires an intact P-loop for its role in these events. Cos2 ATPase activity may be inhibited in cells receiving very low levels of Hh, preventing Ci proteolysis and stabilizing CiFL. The stabilization of CiFL results in the activation of dpp. Nearer the AP border, where higher levels of Hh are received, Su(fu) becomes phosphorylated, inactivating its negative regulatory hold on Ci, while inhibition of the ATPase activity of Cos2 continues to allow stabilization of Ci. In this situation, ptc and dpp are transcribed. Finally, at the highest levels of Hh signaling adjacent to the AP border, Cos2 is required for activation of the pathway and the expression of en. S182N expression, or cos2 over-expression, inhibits the induction of en by endogenous Hh in these cells. The elements of this model are addressed below (Ho, 2005).

Ci plays a central role in determining which genes are repressed or activated in response to different concentrations of Hh. In order to activate target genes such as dpp or ptc, Ci must be stabilized in its full-length form. In wild-type discs, Hh stabilizes Ci by antagonizing molecular events that reduce the concentration of nuclear CiFL. In addition to the constitutive nuclear export of Ci, there are two ways CiFL concentration is reduced: full-length Ci is proteolytically processed into a repressor form; and CiFL is degraded by a lysosome-mediated process involving a novel protein called Debra. In these experiments, the stabilization of CiFL was accomplished by expressing S182N in responsive cells, which antagonizes Cos2 repressor activity and results in the accumulation of high levels of CiFL, with minimal effects on the levels of CiR. This same type of differential effect on CiR and CiFL is accomplished by Debra, which causes the lysosomal degradation of CiFL without affecting the production of CiR. Cos2 and Debra may act in concert to destabilize CiFL, while Cos2 may also aid in the production of CiR via a Debra-independent mechanism. This would involve presenting Ci to the kinases, PKA, CKI and GSKß (Shaggy) for phosphorylation and processing by the proteasome. Since Debra regulates Ci stability in limited areas of the wing disc but S182N can stabilize Ci throughout the anterior compartment, it is likely that S182N interferes with both Debra-dependent and Debra-independent mechanisms of Ci stability to achieve the observed effect: cell-autonomous stabilization of CiFL leading to derepression of dpp (Ho, 2005).

These results suggest that Cos2 may use its ATPase activity to transport Ci to a location where it becomes phosphorylated in preparation for processing, or to the site of processing itself. Alternatively, the ATPase activity may be important for regulating the conformation of Cos2 and its binding to partners such as Smo, Su(fu), Fu and Ci, which would be a novel role for the P-loop in a kinesin-related protein. The S182N mutation may lock Cos2 in a conformation that changes association with binding partners. For example, S182N may decrease the ability of Cos2 to bind Ci, releasing Ci from the cytoplasm, resulting in an increased level of CiFL in the nucleus and the activation of dpp (Ho, 2005).

The human ortholog of Suppressor of fused is a tumor suppressor gene. Su(fu) can associate with Ci, and with the mammalian homologs of Ci, the Gli proteins, through specific protein-protein interactions. Through these interactions, Su(fu) controls the nuclear shuttling of Ci and Gli, as well as the protein stability of CiFL and CiR. Flies homozygous for Su(fu) loss-of-function mutations are normal, so the importance of Su(fu) becomes evident only when other gene functions are thrown out of balance, as in a fu mutant background, with extra or diminished Hh signaling caused by ptc, slimb and protein kinase A mutations or when altered Cos2 is produced (Ho, 2005).

To activate ptc transcription in the wing disc, two conditions have to be met simultaneously: CiFL must be stabilized, and the activity of Su(fu) must be reduced. Removal of Su(fu) changes S182N from a ptc repressor into a ptc activator. Removal of Su(fu) may result in the modification, activation or relocalization of CiFL, or in further sensitizing the system to stabilized CiFL. In Su(fu) homozygous animals, the quantity of CiFL and CiR proteins is greatly diminished, and Su(fu) mutant cells are more sensitized to the Hh signal. The lower levels of both CiFL and CiR in mutant Su(fu) cells may contribute to the sensitivity of these cells to Hh, since a small Hh-driven change in the absolute concentration of either form of Ci would result in a significant change in the ratio between the two proteins. Both CiFL and CiR bind the same enhancer sites, so their relative ratio is likely to be important in determining target gene expression. S182N expression tips the ratio of CiFL to CiR toward CiFL, and reducing the absolute quantities of both Ci isoforms by removing Su(fu) will enhance this effect. Furthermore, Su(fu) binds Ci and sequesters it in the cytoplasm in a stoichiometric manner Reducing the amount of Su(fu) should release more CiFL to the nucleus to activate ptc (Ho, 2005).

The activity of Su(fu) must be regulated or overcome so that target genes can be activated at the right times and places in response to Hh. The regulation of Su(fu) activity may occur by Hh-dependent phosphorylation. A phosphoisoform of Su(fu), Su(fu)-P, was detected in discs where GAL4 was used to drive extra Hh expression. At high concentrations of Hh, the phosphorylation of Su(fu) is not antagonized by overexpression of cos2 or either of the cos2 mutants, suggesting that phosphorylation of Su(fu) occurs independently of Cos2 function. One kinase involved in the phosphorylation of Su(fu) is the Ser/Thr kinase Fused, a well-established component of Hh signal transduction. It is not known whether the phosphorylation of Su(fu) by Fu is direct or indirect (Ho, 2005).

The phosphorylation state of Su(fu) may be an important factor in determining Hh target gene activity. Phosphorylation of an increasing number of Su(fu) molecules with increasing Hh signal may gradually release Ci from all of the known modes of Su(fu)-dependent inhibition, such as nuclear export and recruitment of repressors to nuclear Ci, leading to higher levels of CiFL in the nucleus and the activation of Hh target genes such as ptc (Ho, 2005).

Anterior en expression was used as an in vivo reporter of high levels of Hh signaling. cos2 mutant cells at the AP boundary fail to activate en, suggesting that Cos2 plays a positive regulatory role in en regulation. S182N, S182T and Cos2 overexpression mimics the cos2 loss-of-function condition with respect to en: en remains off in these cells. One interpretation of these data is that all the Cos2 proteins are able to associate with another pathway component, such as Smo, and overproduction of any of them inactivates some of the Smo in non-productive complexes not capable of activating en (Ho, 2005).

In contrast to the activity of all the other mutations generated, deletion of the C terminal domain creates a protein (Cos2DeltaC) that represses normal dpp, ptc and en expression in the wing disc. In this in vivo assay, Cos2DeltaC acts just like wild-type Cos2. A similar deletion has been shown to retain function in cell culture assays. This mutant, expressed under the control of its endogenous promoter, rescues the lethality and wing duplication phenotypes of a cos2 loss-of-function allele over a cos2 deficiency. The results of the rescue experiment bring up a new possibility: that the C-terminal domain of Cos2, and the Cos2-Smo interaction via the C terminus of Cos2, is not necessary for repressor activities of Cos2. Alternatively, Cos2DeltaC could complement or boost the activity of the hypomorphic allele cos211, which was used for the rescue experiment (Ho, 2005).

Targets of Activity

Unrestricted expression of ptc from a heat-shock promoter has no adverse effect on development of Drosophila embryos. The heat-shock construct can also rescue ptc mutants, restoring wg expression to its normal narrow stripe. The ectopic en stripe fails to appear, but the normal one remains unaffected. Despite its localized requirement, the restricted expression of ptc does not itself appear to allocate positional information (Sampedro, 1991). Thus the role of patched in positional signaling is permissive rather than instructive, its activity being required to suppress wingless transcription in cells predisposed to express the latter. According to this view, expression of wingless is normally maintained only in those cells receiving an extrinsic signal, encoded by hedgehog, one that antagonizes the repressive activity of Patched (Ingham, 1991).

In the posterior half of each parasegment Patched protein represses transcription of the wingless gene. In the most posterior row of cells in each parasegment this repression is neutralized by Hedgehog, allowing wg expression. High levels of patched expression might therefore overcome the neutralization by Hedgehog and repress wg in all cells. Transient overexpression of patched in all cells has little or no effect on the segmental pattern. Repeated pulses of patched production drastically alter the segment pattern to mimic embryos lacking wg. Repeated overexpression results in repression of wg and transcription of gooseberry. Expression of two other segment polarity genes, engrailed and cubitus interruptus, is unaffected. Thus excess patched is capable of overcoming the neutralizing signal carried by hedgehog (Schuske, 1994).

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).

Patched inhibits decapentaplegic expression in the anterior compartment of imaginal discs. This results in a restricted expression of dpp near the anterior-posterior compartment boundary, an event which is essential to maintain the wild-type morphology of the wing disc. Viable mutations in the segment polarity genes patched and costal-2 (cos2) cause specific alterations in dpp expression within the anterior compartment of the wing imaginal disc. (Capdevila, 1994b). patched overexpression inhibits transcription of patched and dpp, and post-transcriptionally decreases the amount of Cubitus interrruptus protein at the anterior/posterior boundary (Johnson, 1995).

Patched regulates wingless in an early hh-independent phase and also in a late hh-independent phase. Direct WG autoregulation (autocrine signaling) 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 signaling. Direct WG autoregulation differs from WG signaling to adjacent cells in the importance of fused, smoothened and cubitus interruptus relative to zw3 and armadillo. WG autoregulation during this early hh-dependent phase differs from later WG autoregulation in the lack of gooseberry participation. (Hooper, 1994).

Patched targets gooseberry distal and gooseberry-proximal in neuroblast determination. The RP2 neuron is a motoneuron and innervates muscle number 2 of the dorsal musculature. This neuron originates along with its sibling cell from the first ganglion mother cell derived from NB4-2, and occupies the anterior commissure along with several other RP2 neurons. NB4-2 itself is formed during the second wave of neuroblast delamination in stage 9. Gooseberry and Patched participate in the Wingless-mediated specification of NB4-2 by controlling the response to the wingless signal. In gsb mutants, WG-positive NB5-3 is transformed to NB4-2 in a Wg-dependent manner, suggesting that GSB normally represses the capacity to respond to the wingless signal. In ptc mutants, gsb is ectopically expressed in normally Wg-reponsive cells, thus preventing the response to Wingless and consequently the correct specification of NB4-2 does not take place. The timing of the response to GSB suggests that the specification of neuroblast identities takes place within the neuroectoderm, prior to neuroblast delamination (Bhat, 1996).

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

A positive role for Patched in Hedgehog signaling revealed by the intracellular trafficking of Sex-lethal, the Drosophila sex determination master switch

The sex determination master switch, Sex-lethal (Sxl), controls sexual development as a splicing and translational regulator. Hedgehog (Hh) is a secreted protein that specifies cell fate during development. Sxl is in a complex that contains all of the known Hh cytoplasmic components, including Cubitus interruptus (Ci) the only known target of Hh signaling. Hh promotes the entry of Sxl into the nucleus in the wing disc. In the anterior compartment, the Hh receptor Patched (Ptc) is required for this effect, revealing Ptc as a positive effector of Hh. Some of the downstream components of the Hh signaling pathway also alter the rate of Sxl nuclear entry. Mutations in Suppressor of Fused or Fused with altered ability to anchor Ci are also impaired in anchoring Sxl in the cytoplasm. The levels, and consequently, the ability of Sxl to translationally repress downstream targets in the sex determination pathway, can also be adversely affected by mutations in Hh signaling genes. Conversely, overexpression of Sxl in the domain that Hh patterns negatively affects wing patterning. These data suggest that the Hh pathway impacts on the sex determination process and vice versa and that the pathway may serve more functions than the regulation of Ci (Horabin, 2003).

Sxl co-immunoprecipitates with Cos2 and Fu in the female germline. Since Ci is not expressed in germ cells, it is probable that a different Hh cytoplasmic complex might exist in germ cells. In somatic cells, Sxl is expressed in all female cells while Ci is expressed in only a subset. To test whether the Hh pathway differentiates between the two proteins in somatic cells, Sxl was immunoprecipitated from embryonic extracts and the immunoprecipitates probed for the various Hh cytoplasmic components. The immunoprecipitates showed that Cos2, Fu and Ci are complexed with Sxl. The specificity of this association of Sxl with the Hh pathway components was verified using antibodies to either Ci or Su(fu), and testing the immunoprecipitates for the presence of Sxl. Both co-immunoprecipitated with Sxl. The Ci immunoprecipitate was also tested for another Hh cytoplasmic component, Fu, which was present as expected. These interactions are maintained in a Su(fu)LP background (protein null allele). An IP of Ci from Su(fu)LP embryos brought down Sxl, as well as Fu and Cos2. Taken together, these data suggest that cells that express Ci and Sxl have both proteins in the same complex with the known cytoplasmic components of the Hh signaling pathway (Horabin, 2003).

Previous work on the germline has suggested that the Hh signaling pathway affects the intracellular trafficking Sxl. The cross talk between these two developmental pathways has been analyzed in tissues where both Hh targets can be present in the same cell. While analysis of embryos only uncovered an effect of Cos2 on Sxl, analysis of wing discs allowed several specific effects to be uncovered. At least three new functional aspects of the Hh pathway are suggested:

  1. More than one 'target' protein can exist in the Hh cytoplasmic complex.
    Immunoprecipitation experiments using extracts from embryos indicate that Sex-lethal and the known Hh signaling target Ci are in the same complex. The two proteins can co-immunoprecipitate each other as well as other known members of the Hh cytoplasmic complex. Even when Su(fu), the cytoplasmic component that most strongly anchors Sxl in the cytoplasm, is removed, Sxl can still be co-immunoprecipitated with both Ci and Fu. As a whole, these results suggest that at least some proportion of the two Hh 'target' proteins are in a common complex within the cell. Additionally, the wing defects produced when Sxl is overexpressed in the Hh signaling region suggest that their relative concentrations are important for their normal functioning (Horabin, 2003).
  2. The Hh targets can be affected differentially.
    The presence of two 'targets' within the Hh cytoplasmic complex, raises the question of how they can be differentially affected. The data show that the various members of the Hh pathway do not affect Sx1 and Ci similarly. Smo appears to be dispensable for the transmission of the Hh signal in promoting Sx1 nuclear entry, while Smo is critical for the activation of Ci. Conversely, while Ptc is essential for the effect of Hh on Sxl, it is dispensable for the activation of Ci. The Fu kinase (fumH63 background) also appears to have no role in Hh signaling with respect to Sxl, while it is critical for the activation of Ci. By contrast, both Su(fu) and the Fu regulatory domain act similarly on Sxl and Ci, serving to anchor them in the cytoplasm (Horabin, 2003).

    Taken together, these data suggest that the presence of Hh can be relayed to the cytoplasmic components differentially and, while the data do not address the point, suggest how different outcomes might be achieved. Ptc has been proposed to be a transmembrane transporter protein that functions catalytically in the inhibition of Smo via a diffusible small molecule. The stimulation of Sxl nuclear entry by the binding of Hh to Ptc might also involve a change in the internal cell milieu, but in this case the Hh cytoplasmic complex may be affected independently, not requiring a change in the activity of Smo or the Fu kinase (Horabin, 2003).

  3. Ptc can signal the presence of the Hh ligand in a positive manner.
    Several experiments indicate that Hh bound to Ptc enhances the nuclear entry of Sxl. That Smo has no role in transmitting the Hh signal is most clearly demonstrated by expressing the PtcD584 protein in both the anterior and posterior compartments of the dorsal half of the wing disc. PtcD584 acts as a dominant negative and so activates Ci in the anterior compartment, but it fails to enhance the levels of nuclear Sxl in the anterior because it sequesters Hh in the posterior compartment. The double mutant condition of ptc clones in a hhMRT background clearly places Ptc downstream of Hh, while showing Ptc can act positively in transmitting the Hh signal (Horabin, 2003).

A positive role for Ptc, but in this case in conjunction with Smo, in promoting cell proliferation during head development has recently been reported. In this situation, however, Hh acts negatively on both Ptc and Smo in their activation of the Activin type I receptor, suggesting an even greater variance from the canonical Hh signaling process (Horabin, 2003).

While the effects on Sxl in the anterior compartment show a dependence on the known Hh signaling components, it is not clear what promotes the rapid nuclear entry of Sxl in the posterior compartment. Su(fu) is expressed uniformly across the disc so it does not appear to be responsible for the AP differences, and ptc clones have no effect (and Ptc RNA and protein are not detected in the posterior compartment). Removal of Hh, however, reduces the nuclear entry rate of Sxl in both compartments. In this regard, the parallel between Hh pathway activation and Sxl nuclear entry in the posterior compartment is worth noting. Fu is also activated in the posterior compartment in a Hh-dependent manner, even though Ptc is not present. It is not clear what mediates between Hh and Fu (Horabin, 2003).

The data also suggest that the Hh cytoplasmic complex may have slightly different compositions in different tissues and/or at developmental stages. In the female germline and in embryos, the absence of Cos2 leads to a severe reduction in Sxl levels. However, in wing discs when mutant clones are made using the same cos2 allele, there is no effect on Sxl. It is suggested that between the third instar larval stage and eclosion, the composition of the Hh cytoplasmic complex may change again to make Sxl more sensitive to Cos2. This would explain why removal of Cos2 can produce sex transformations of the foreleg even though mutant clones in wing discs (and also leg discs) show no alterations in Sxl levels (Horabin, 2003).

A similar argument might apply to the weak sex transformations of forelegs produced by PKA clones. Alternatively, PKA may have a very weak effect but the assay on wing discs is not sufficiently sensitive to allow detection of small effects; PKA was found to have a modest effect on Sxl nuclear entry in the germline. Sxl is sufficiently small (38-40 kDa) to freely diffuse into the nucleus, or the protein may enter the nucleus as a complex with splicing components. This may account for the limited sex transformations caused by removal of Hh pathway components (Horabin, 2003).

Removal of several of the Hh pathway components, such as smo, gives the same weak sex transformation phenotype, even though smo has no effect on Sxl nuclear entry. Additionally, there is no correlation between a positive and a negative Hh signaling component and whether there is a resulting phenotype. Changing the dynamics of the activation state of the Hh cytoplasmic complex may perturb the normal functioning of Sxl, since Sxl appears to be in the same complex as Ci. For example, if the Hh pathway is fully activated because of a mutant condition, the relative amounts of Sxl in the cytoplasm versus nucleus at any given time, may be different from the wild-type condition. Perturbing the usual cytoplasmic-nuclear balance could compromise the various processes that Sxl protein regulates. Sxl acts both positively and negatively on its own expression through splicing and translation control and, additionally, regulates the downstream sex differentiation targets. The latter could also be responsible for the weak sex transformations seen, in view of the recent demonstration that doublesex affects the AP organizer and sex-specific growth in the genital disc (Horabin, 2003).

With the exception of Cos2, which can produce relatively substantial effects on Sxl levels in embryos as well as sex transformations in the foreleg, the effects of removal of any of the other Hh pathway components are generally not large. The strong effects of Cos2 on Sxl could be because it affects the stability of Sxl. However, Sxl depends on an autoregulatory splicing feedback loop for its maintenance making the protein susceptible to a variety of regulatory breakdowns. If Cos2 altered the nuclear entry of Sxl, for example, its removal could compromise the female-specific splicing of Sxl transcripts by reducing the amounts of nuclear Sxl. Splicing of Sxl transcripts would progressively fall into the male mode to eventually result in a loss of Sxl protein (Horabin, 2003).

Cos2 and Fu have been reported to shuttle into and out of the nucleus, and their rate of nuclear entry is not dependent on the Hh signal. That Ci and Sxl are complexed with the same Hh pathway cytoplasmic components, and share and yet have unique intracellular trafficking responses to mutations in the pathway, makes it tempting to speculate that the Hh cytoplasmic components may have had a functional origin related to intracellular trafficking that preceded the two proteins. Whether this reflects a more expanded role in regulated nuclear entry remains to be determined (Horabin, 2003).

Sxl elevates ptc expression

Sex-lethal (Sxl), the Drosophila sex-determination master switch, is on in females and controls sexual development as a splicing and translational regulator. Hedgehog (Hh) is a secreted protein that specifies cell fate during development. Sxl protein has been shown to be part of the Hh cytoplasmic signaling complex and Hh promotes Sxl nuclear entry. In the wing disc anterior compartment, Patched (Ptc), the Hh receptor, acts positively in this process. This study shows that the levels and rate of nuclear entry of full-length Cubitus interruptus (Ci), the Hh signaling target, are enhanced by Sxl. This effect requires the cholesterol but not palmitoyl modification on Hh, and expands the zone of full-length Ci expression. Expansion of Ci activation and its downstream targets, particularly decapentaplegic the Drosophila TGFß homolog, suggests a mechanism for generating different body sizes in the sexes; in Drosophila, females are larger and this difference is controlled by Sxl. Consistent with this proposal, discs expressing ectopic Sxl show an increase in growth. In keeping with the idea of the involvement of a signaling system, this growth effect by Sxl is not cell autonomous. These results have implications for all organisms that are sexually dimorphic and use Hh for patterning (Horabin, 2005) (Horabin, 2005).

In both sexes, ectopic expression of Sxl shows an increase in intensity of ptc expression, indicating it is possible to further elevate the Hh response. Other than en, which was difficult to score in these experiments, ptc requires the highest levels of Ci activation for its transcription (Horabin, 2005).

In females, the ectopic Sxl elevates ptc expression in the cells near the AP boundary, but the depth of the cells showing this highest level of Ci activation is reduced. A reduction in the number of cells transcribing ptc, when compared with the wider but less intense width of ptc transcription in the control half of the disc, suggests a restriction in Hh diffusion. Elevated ptc transcription is expected to produce more Ptc at the membrane, which should sequester more Hh close to the AP boundary. This result shows that Sxl can both enhance the Hh response and effectively alter the Hh gradient (Horabin, 2005).

In males, the increase in ptc transcription induced by Sxl both intensifies and widens the ptc expression zone. This suggests that the activation of Ci is at a lower peak in males than in females, and its enhancement by ectopic Sxl does not reach the same maximum that additional Sxl in females produces (Horabin, 2005).

The Hh pathway can also control body size in mammals. ptc1 mutations in mice provide an overgrowth phenotype with large body size, while increasing ptc1 expression decreases body size. Humans with basal cell nevus syndrome, an autosomal-dominant condition caused by the inheritance of a mutant ptc allele, have been reported to have multiple developmental abnormalities and, relevant to this study, larger body size. Whether the mechanism described in this study is global to sexually dimorphic organisms that use Hh for patterning remains to be seen (Horabin, 2005).

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

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