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Wnt proteins are secreted, lipid-modified glycoproteins that control animal development and adult tissue homeostasis. Secretion of Wnt proteins is at least partly regulated by a dedicated machinery. Here, we report a genome-wide RNA interference screen for genes involved in the secretion of Wingless (Wg), a Drosophila Wnt. We identify three new genes required for Wg secretion. Of these, Emp24 and Eclair are required for proper export of Wg from the endoplasmic reticulum (ER). We propose that Emp24 and Eca act as specific cargo receptors for Wg to concentrate it in forming vesicles at sites of ER export.

The Hedgehog (Hh) pathway, an evolutionarily conserved key regulator of embryonic patterning and tissue homeostasis, controls its target genes by managing the processing and activities of the Gli/Ci transcription factors. Little is known about the nuclear co-factors the Gli/Ci proteins recruit, and how they mechanistically control Hh target genes. Here, we provide evidence for the involvement of Parafibromin/Hyx as a positive component in Hh signaling. We found that hyx RNAi impaired Hh pathway activity in Drosophila cell culture. Consistent with an evolutionarily conserved function in Hh signaling, RNAi-mediated knockdown of Parafibromin in mammalian cell culture experiments diminished the transcriptional activity of Gli1 and Gli2. In vivo , in Drosophila , genetic impairment of hyx decreased the expression of the high-threshold Hh target gene knot / collier . Conversely, hyx overexpression ameliorated the inhibitory activity of Ptc and Ci 75 misexpression during Drosophila wing development. We subsequently found that Parafibromin can form a complex with all three Glis, and provide evidence that Parafibromin/Hyx directly binds Region 1, the Su(fu) interaction domain, in the N-terminus of all Glis and Ci. Taken together, our results suggest a target gene-selective involvement of the PAF1 complex in Hh signaling via the Parafibromin/Hyx-mediated recruitment to Gli/Ci.

Wnt ligands are lipid-modified, secreted glycoproteins that control multiple steps during embryogenesis and adult-tissue homeostasis. Little is known about the mechanisms underlying Wnt secretion. Recently, Wntless (Wls/Evi/Srt) was identified as a conserved multi-pass transmembrane protein whose function seems to be dedicated to promoting the release of Wnts. Here, we describe Wls accumulation in the Golgi apparatus of Wnt/Wingless (Wg)-producing cells in Drosophila, and show that this localization is essential for Wg secretion. Moreover, Wls localization and levels critically depend on retromer, a conserved protein complex that mediates endosome-to-Golgi protein trafficking in yeast. In the absence of the retromer components Dvps35 or Dvps26, but in presence of Wg, Wls is degraded and Wg secretion impaired. Our results indicate that Wg, clathrin-mediated endocytosis and retromer sustain a Wls traffic loop from the Golgi to the plasma membrane and back to the Golgi, thereby enabling Wls to direct Wnt secretion.

How functional WNT proteins are made and how their secretion is regulated is becoming a focal point for the WNT-signalling field. Recently, lipoprotein particles, WNT lipid modifications, the conserved transmembrane protein Wntless (WLS; also known as EVI and SRT) and the retromer complex have been implicated in WNT secretion. Our aim is to synthesize ideas from these new findings for the mechanisms that underlie WNT secretion.

Developing tissues that contain mutant or compromised cells present risks to animal health. Accordingly, the appearance of a population of suboptimal cells in a tissue elicits cellular interactions that prevent their contribution to the adult. Here we report that this quality control process, cell competition, uses specific components of the evolutionarily ancient and conserved innate immune system to eliminate Drosophila cells perceived as unfit. We find that Toll-related receptors (TRRs) and the cytokine Spätzle (Spz) lead to NFκB-dependent apoptosis. Diverse “loser” cells require different TRRs and NFκB factors and activate distinct pro-death genes, implying that the particular response is stipulated by the competitive context. Our findings demonstrate a functional repurposing of components of TRRs and NFκB signaling modules in the surveillance of cell fitness during development.

The wing imaginal disc is subdivided into two nonintermingling sets of cells, the anterior (A) and posterior (P) compartments. Anterior cells require reception of the Hedgehog (Hh) signal to segregate from P cells. We provide evidence that Hh signaling controls A/P cell segregation not by directly modifying structural components but by a Cubitus interruptus (Ci)-mediated transcriptional response. A shift in the balance between repressor and activator forms of Ci toward the activator form is necessary and sufficient to define “A-type” cell sorting behavior. Moreover, we show that Engrailed (En), in the absence of Ci, is sufficient to specify “P-type” sorting. We propose that the opposing transcriptional activities of Ci and En control cell segregation at the A/P boundary by regulating a single cell adhesion molecule.

Wartlick et al . (Research Articles, 4 March 2011, p. 1154) reported that growth rates in the Drosophila wing disc correlate with increasing Dpp signaling levels, suggesting that the rate of Dpp increase determines the cell-cycle length. Contradicting their model, we found that cells in which the increase of Dpp signaling levels was genetically abrogated grew at rates comparable to those of wild-type cells.

β-Catenin, apart from playing a cell-adhesive role, is a key nuclear effector of Wnt signaling. Based on activity assays in Drosophila, we generated mouse strains where the endogenous β-catenin protein is replaced by mutant forms, which retain the cell adhesion function but lack either or both of the N- and the C-terminal transcriptional outputs. The C-terminal activity is essential for mesoderm formation and proper gastrulation, whereas N-terminal outputs are required later during embryonic development. By combining the double-mutant β-catenin with a conditional null allele and a Wnt1-Cre driver, we probed the role of Wnt/β-catenin signaling in dorsal neural tube development. While loss of β-catenin protein in the neural tube results in severe cell adhesion defects, the morphology of cells and tissues expressing the double-mutant form is normal. Surprisingly, Wnt/β-catenin signaling activity only moderately regulates cell proliferation, but is crucial for maintaining neural progenitor identity and for neuronal differentiation in the dorsal spinal cord. Our model animals thus allow dissecting signaling and structural functions of β-catenin in vivo and provide the first genetic tool to generate cells and tissues that entirely and exclusively lack canonical Wnt pathway activity.

We use the Dpp morphogen gradient in the Drosophila wing disc as a model to address the fundamental question of how a gradient of a growth factor can produce uniform growth. We first show that proper expression and subcellular localization of components in the Fat tumor-suppressor pathway, which have been argued to depend on Dpp activity differences, are not reliant on the Dpp gradient. We next analyzed cell proliferation in discs with uniformly high Dpp or uniformly low Fat signaling activity and found that these pathways regulate growth in a complementary manner. While the Dpp mediator Brinker inhibits growth in the primordium primarily in the lateral regions, Fat represses growth mostly in the medial region. Together, our results indicate that the activities of both signaling pathways are regulated in a parallel rather than sequential manner and that uniform proliferation is achieved by their complementary action on growth.

Wingless (Wg) signaling regulates expression of its target genes via Pangolin and Armadillo, and their interacting cofactors. In the absence of Wg, Pangolin mediates transcriptional repression. In the presence of Wg, Pangolin, Armadillo, and a cohort of coactivators mediate transcriptional activation. Here we uncover Coop (corepressor of Pan) as a Pangolin-interacting protein. Coop and Pangolin form a complex on DNA containing a Pangolin/TCF-binding motif. Overexpression of Coop specifically represses Wg target genes, while loss of Coop function causes derepression. Finally, we show that Coop antagonizes the binding of Armadillo to Pangolin, providing a mechanism for Coop-mediated repression of Wg target gene transcription.

Apical cell surfaces in metazoan epithelia, such as the wing disc of Drosophila, resemble polygons with different numbers of neighboring cells. The distribution of these polygon numbers has been shown to be conserved. Revealing the mechanisms that lead to this topology might yield insights into how the structural integrity of epithelial tissues is maintained. It has previously been proposed that cell division alone, or cell division in combination with cell rearrangements, is sufficient to explain the observed epithelial topology. Here, we extend this work by including an analysis of the clustering and the polygon distribution of mitotic cells. In addition, we study possible effects of cellular growth regulation by mechanical forces, as such regulation has been proposed to be involved in wing disc size regulation. We formulated several theoretical scenarios that differ with respect to whether cell rearrangements are allowed and whether cellular growth rates are dependent on mechanical stress. We then compared these scenarios with experimental data on the polygon distribution of the entire cell population, that of mitotic cells, as well as with data on mitotic clustering. Surprisingly, we observed considerably less clustering in our experiments than has been reported previously. Only scenarios that include mechanical-stress-dependent growth rates are in agreement with the experimental data. Interestingly, simulations of these scenarios showed a large decrease in rearrangements and elimination of cells. Thus, a possible growth regulation by mechanical force could have a function in releasing the mechanical stress that evolves when all cells have similar growth rates.

The use of binary transcriptional systems offers many advantages for experimentally manipulating gene activity, as exemplified by the success of the Gal4/UAS system in Drosophila . To expand the number of applications, a second independent transactivator (TA) is desirable. Here, we present the optimization of an additional system based on LexA and show how it can be applied. We developed a series of LexA TAs, selectively suppressible via Gal80, that exhibit high transcriptional activity and low detrimental effects when expressed in vivo. In combination with Gal4, an appropriately selected LexA TA permits to program cells with a distinct balance and independent outputs of the two TAs. We demonstrate how the two systems can be combined for manipulating communicating cell populations, converting transient tissue-specific expression patterns into heritable, constitutive activities, and defining cell territories by intersecting TA expression domains. Finally, we describe a versatile enhancer trap system that allows swapping TA and generating mosaics composed of Gal4 and LexA TA-expressing cells. The optimized LexA system facilitates precise analyses of complex biological phenomena and signaling pathways in Drosophila .

SummaryDi- and trimethylation of histone H3 lysine 4 (H3K4me2 and H3K4me3) are hallmarks of chromatin at active genes [1]. The major fraction of K4-methylated histone H3 is the variant H3 (termed H3.3 in Drosophila) [2], which replaces canonical H3 (H3.2) in transcribed genes [3, 4]. Here, we genetically address the in vivo significance of such K4 methylation by replacing wild-type H3.3 with a mutant form (H3.3K4A) that cannot be methylated. We monitored the transcription that occurs in response to multiple well-described signaling pathways. Surprisingly, the transcriptional outputs of these pathways remain intact in H3.3K4A mutant cells. Even the complete absence of both H3.3 genes does not noticeably affect viability or function of cells: double mutant animals are viable but sterile. Fertility can be rescued by K4-containing versions of H3.3, but not with mutant H3.3 (H3.3K4A) or with canonical H3.2. Together, these data suggest that in Drosophila, presence of H3.3K4me in the chromatin of active genes is dispensable for successful transcription in most cells and only plays an important role in reproductive tissues.

Morphogens can control organ development by regulating patterning as well as growth. Here we use the model system of the Drosophila wing imaginal disc to address how the patterning signal Decapentaplegic (Dpp)regulates cell proliferation. Contrary to previous models, which implicated the slope of the Dpp gradient as an essential driver of cell proliferation, we find that the juxtaposition of cells with differential pathway activity is not required for proliferation. Additionally, our results demonstrate that, as is the case for patterning, Dpp controls wing growth entirely via repression of the target gene brinker (brk). The Dpp-Brk system converts an inherently uneven growth program, with excessive cell proliferation in lateral regions and low proliferation in medial regions, into a spatially homogeneous profile of cell divisions throughout the disc.

SummaryCell-cell communication via Wnt signals represents a fundamental means by which animal development and homeostasis are controlled. The identification of components of the Wnt pathway is reaching saturation for the transduction process in receiving cells but is incomplete concerning the events occurring in Wnt-secreting cells. Here, we describe the discovery of a novel Wnt pathway component, Wntless (Wls/Evi), and show that it is required for Wingless-dependent patterning processes in Drosophila, for MOM-2-governed polarization of blastomeres in C. elegans, and for Wnt3a-mediated communication between cultured human cells. In each of these cases, Wls is acting in the Wnt-sending cells to promote the secretion of Wnt proteins. Since loss of Wls function has no effect on other signaling pathways yet appears to impede all the Wnt signals we analyzed, we propose that Wls represents an ancient partner for Wnts dedicated to promoting their secretion into the extracellular milieu.

Members of the Wingless (Wg)/Wnt family of secreted glycoproteins control cell fate during embryonic development and adult homeostasis. Wnt signals regulate the expression of target genes by activating a conserved signal transduction pathway. Upon receptor activation, the signal is transmitted intracellularly by stabilization of Armadillo (Arm)/beta-catenin. Arm/beta-catenin translocates to the nucleus, interacts with DNA-binding factors of the Pangolin (Pan)/TCF/LEF class and activates transcription of target genes in cooperation with the recently identified proteins Legless/BCL9 (Lgs) and Pygopus (Pygo). Here, we analyse the mode of action of Pan, Arm, Lgs, and Pygo in Drosophila cultured cells. We provide evidence that together these four proteins form a 'chain of adaptors' linking the NH2-terminal homology domain (NHD) of Pygo to the DNA-binding domain of Pan. We show that the NHD has potent transcriptional activation capacity, which differs from that of acidic activator domains and depends on a conserved NPF tripeptide. A single point mutation within this NPF motif abolishes the transcriptional activity of the Pygo NHD in vitro and strongly reduces Wg signalling in vivo. Together, our results suggest that the transcriptional output of Wg pathway activity largely relies on a 'chain of adaptors' design to direct the Pygo NHD to Wg target promoters in an Arm-dependent manner.

The Wnt signalling system controls many fundamental processes during animal development and its deregulation has been causally linked to colorectal cancer. Transduction of Wnt signals entails the association of beta-catenin with nuclear TCF DNA-binding factors and the subsequent activation of target genes. Using genetic assays in Drosophila, we have recently identified a presumptive adaptor protein, Legless (Lgs), that binds to beta-catenin and mediates signalling activity by recruiting the transcriptional activator Pygopus (Pygo). Here, we characterize the beta-catenin/Lgs interaction and show: (1) that it is critically dependent on two acidic amino acid residues in the first Armadillo repeat of beta-catenin; (2) that it is spatially and functionally separable from the binding sites for TCF factors, APC and E-cadherin; (3) that it is required in endogenous as well as constitutively active forms of beta-catenin for Wingless signalling output in Drosophila; and (4) that in its absence animals develop with the same phenotypic consequences as animals lacking Lgs altogether. Based on these findings, and because Lgs and Pygo have human homologues that can substitute for their Drosophila counterparts, we infer that the beta-catenin/Lgs binding site may thus serve as an attractive drug target for therapeutic intervention in beta-catenin-dependent cancer progression.

Overexpression of myc protooncogenes has been implicated in the genesis of many human tumors. Myc proteins seem to regulate diverse biological processes, but their role in tumorigenesis remains enigmatic. Here we use Drosophila imaginal discs to mimic situations in which cells with unequal levels of Myc protein are apposed and show that this invariably elicits a win/lose situation reminiscent of cell competition; cells with lower levels of dMyc are eliminated by apoptosis whereas cells with higher levels of dMyc overproliferate. We find that this competitive behavior correlates with, and can be corrected by, the activation of the BMP/Dpp survival signaling pathway. Hence the heritable increase in dMyc levels causes cells to behave as “super-competitors” and reveals a novel mode of clonal expansion that causes, but also relies on, the killing of surrounding cells.

The development of multicellular organisms requires the establishment of cell populations with different adhesion properties. In Drosophila, a cell-segregation mechanism underlies the maintenance of the anterior (A) and posterior (P) compartments of the wing imaginal disc. Although engrailed (en) activity contributes to the specification of the differential cell affinity between A and P cells, recent evidence suggests that cell sorting depends largely on the transduction of the Hh signal in A cells. The activator form of Cubitus interruptus (Ci), a transcription factor mediating Hh signaling, defines anterior specificity, indicating that Hh-dependent cell sorting requires Hh target gene expression. However, the identity of the gene(s) contributing to distinct A and P cell affinities is unknown. Here, we report a genetic screen based on the FRT/FLP system to search for genes involved in the correct establishment of the anteroposterior compartment boundary. By using double FRT chromosomes in combination with a wing-specific FLP source we screened 250,000 mutagenized chromosomes. Several complementation groups affecting wing patterning have been isolated, including new alleles of most known Hh-signaling components. Among these, we identified a class of patched (ptc) alleles exhibiting a novel phenotype. These results demonstrate the value of our setup in the identification of genes involved in distinct wing-patterning processes.

Secreted peptide signals control many fundamental processes during animal development. Proper responses to these signals require cognate inducible feedback antagonists. Here we report the identification of a novel Drosophila Wingless (Wg) target gene, wingful(wf), and show that it encodes a potent extracellular feedback inhibitor of Wg. In contrast to the cytoplasmic protein Naked cuticle (Nkd), the only known Wg feedback antagonist, Wf functions during larval stages, when Nkd function is dispensable. We propose that Wf may provide feedback control for the long-range morphogen activities of Wg.

Here we report the identification of Dfz3, a novel member of the Frizzled family of seven-pass transmembrane receptors. Like Dfz2, Dfz3 is a target gene of Wingless (Wg) signalling, but in contrast to Dfz2, it is activated rather than repressed by Wg signalling in imaginal discs. We show that Dfz3 is not required for viability but is necessary for optimal Wg signalling at the wing margin.

The gradient morphogen Decapentaplegic (Dpp) organizes pattern by inducing the transcription of different target genes at distinct threshold concentrations during Drosophila development. An important, albeit indirect, mode by which Dpp controls the spatial extent of its targets is via the graded downregulation of brinker, whose product in turn negatively regulates the expression of these targets. Here we report the molecular dissection of the cis-regulatory sequences of optomotor-blind (omb), a Dpp target gene in the wing. We identify a minimal 284 bp Dpp response element and demonstrate that it is subject to Brinker (Brk) repression. Using this omb wing enhancer, we show that Brk is a sequence-specific DNA binding protein. Mutations in the high-affinity Brk binding site abolish responsiveness of this omb enhancer to Brk and also compromise the input of an unknown transcriptional activator. Our results therefore identify Brk as a novel transcription factor antagonizing Dpp signalling by directly binding target genes and repressing their expression.

Members of the Hedgehog (Hh) family of secreted signaling proteins function as potent short-range organizers in animal development. Their range of action is limited by a C-terminal cholesterol tether and the upregulation of Patched (Ptc) receptor levels. Here we identify a novel segment-polarity gene in Drosophila, dispatched (disp), and demonstrate that its product is required in sending cells for normal Hh function. In the absence of Disp, cholesterol-modified but not cholesterol-free Hh is retained in these cells, indicating that Disp functions to release cholesterol-anchored Hh. Despite their opposite roles, Disp and Ptc share structural homology in the form of a sterol-sensing domain, suggesting that release and sequestration of cholesterol-modified Hh may be based on related molecular pathways.

In both vertebrates and Drosophila, limb development is organized by a posteriorly located source of the signalling protein Hedgehog (Hh) [1] [2] [3] [4]. In Drosophila, the expression of Hh target genes is controlled by two opposing activities of the transcriptional regulator Cubitus interruptus (Ci), which activates target genes in response to Hh signalling but is converted into a repressor form in the absence of Hh [5] [6] [7] [8] [9] [10]. Three homologs of Ci (Gli1, Gli2, and Gli3) have been implicated in mediating responses to Sonic hedgehog (Shh) in vertebrates [11] [12]. Much attention has been devoted to the expression pattern of GLI genes; GLI1 is induced by Shh, whereas GLI3 transcription appears to be repressed by Shh signalling [13] [14] [15]. The regulation of GLI gene expression is therefore one important mechanism by which GLI genes organize pattern. It is not well understood, however, whether Shh signalling also controls the activities of Gli proteins post-translationally and whether these activities have activating or repressing effects on target genes in vivo. Here, we have subjected the human proteins Gli1 and Gli3 to the precise and well-defined Hh signalling assay of Drosophila wing development and established that Gli1 functions as an activator and Gli3 as a repressor of Hh target genes; that the activating transcriptional activity of Gli1 and the repressing activity of Gli3 are both subject to Hh regulation in vivo; and that the combined activities of Gli1 and Gli3 can substitute for Ci in controlling Hh target gene expression during embryonic and larval development.

The Hedgehog (Hh) and Wingless (Wg) signaling pathways play important roles in animal development. The activities of the two pathways depend on each other during Drosophila embryogenesis. In the embryonic segment, Wg is required in anterior cells to sustain Hh secretion in adjacent posterior cells. Hh input in turn is necessary for anterior cells to maintain wg expression. The Hh and Wg pathways are mediated by the transcription factors Cubitus interruptus (Ci) and Pangolin/TCF (Pan), respectively. Coincidentally, pan and ci are adjacent genes on the fourth chromosome in a head-to-head orientation. Our genetic and in situ hybridization data indicate that ci D is a mutation affecting both ci and pan . Molecular analysis revealed that the ci D allele is caused by an inversion event that swapped the promoter regions and the first exons of the two genes. The ci gene in ci D is controlled by the ubiquitous pan promoter and encodes a hybrid Ci protein that carries the N-terminal region of Pan. This domain has previously been shown to bind to the β-catenin homolog Armadillo (Arm), raising the possibility that Wg input, in addition to Hh input, modulates the activity of the hybrid Ci D protein. Indeed, we found that Wg signaling induces the expression of the Hh target gene patched ( ptc ) in ci D animals. We provide evidence that this effect depends on the ability of the Ci D protein to bind Arm. Genetic and molecular data indicate that wild-type Pan and Ci D compete for binding to Arm, leading to a compromised transduction of the Wg signal in heterozygous ci D /+ animals and to a dramatic enhancement of the gain-of-function activity of Ci D in homozygous mutants. Thus, the Hh and the Wg pathways are affected by the ci D mutation, and the Ci D fusion protein integrates the activities of both.

The TGF-β homolog Decapentaplegic (Dpp) acts as a secreted morphogen in the Drosophila wing disc, and spreads through the target tissue in order to form a long range concentration gradient. Despite extensive studies, the mechanism by which the Dpp gradient is formed remains controversial. Two opposing mechanisms have been proposed: receptor-mediated transcytosis (RMT) and restricted extracellular diffusion (RED). In these scenarios the receptor for Dpp plays different roles. In the RMT model it is essential for endocytosis, re-secretion, and thus transport of Dpp, whereas in the RED model it merely modulates Dpp distribution by binding it at the cell surface for internalization and subsequent degradation. Here we analyzed the effect of receptor mutant clones on the Dpp profile in quantitative mathematical models representing transport by either RMT or RED. We then, using novel genetic tools, experimentally monitored the actual Dpp gradient in wing discs containing receptor gain-of-function and loss-of-function clones. Gain-of-function clones reveal that Dpp binds in vivo strongly to the type I receptor Thick veins, but not to the type II receptor Punt. Importantly, results with the loss-of-function clones then refute the RMT model for Dpp gradient formation, while supporting the RED model in which the majority of Dpp is not bound to Thick veins. Together our results show that receptor-mediated transcytosis cannot account for Dpp gradient formation, and support restricted extracellular diffusion as the main mechanism for Dpp dispersal. The properties of this mechanism, in which only a minority of Dpp is receptor-bound, may facilitate long-range distribution., Author Summary Morphogens are signaling molecules that trigger specific responses in cells in a concentration-dependent manner. The formation of morphogen gradients is essential for the patterning of tissues and organs. Decapentaplegic (Dpp) is the Drosophila homolog of the bone morphogenic proteins in vertebrates and forms a morphogen gradient along the anterior-posterior axis of the Drosophila wing imaginal disc, a single-cell layered epithelium. Dpp determines the growth and final size of the wing disc and serves as an ideal model system to study gradient formation. Despite extensive studies the mechanism by which morphogen gradients are established remains controversial. In the case of Dpp two mechanisms have been postulated, namely extracellular diffusion and receptor-mediated transcytosis. In the first model Dpp is suggested to move by diffusion through the extracellular matrix of a tissue, whereas in the latter model Dpp is transported through the cells by receptor-mediated uptake and re-secretion. In this work we combined novel genetic tools with mathematical modeling to discriminate between the two models. Our results suggest that the Dpp gradient forms following the extracellular diffusion mechanism. Moreover, our data suggest that the majority of the extracellular Dpp is free and not bound to its receptor, a property likely to play a role for the long-range gradient formation.

Cell fate in the developing eye is determined by a cascade of inductive interactions. In this process, the sevenless protein--a receptor tyrosine kinase--is required for the specification of the R7 photoreceptor cell fate. We have constructed a gain-of-function sevenless mutation (SevS11) by overexpressing a truncated sevenless protein in the cells where sevenless is normally expressed. In SevS11 mutant flies, all sevenless-expressing cells initiate neural development. This results in the formation of multiple R7-like photoreceptors per ommatidium. Therefore, sevenless activity appears to be necessary and sufficient for the determination of R7 cell fate. These results illustrate the central role receptor tyrosine kinases can play in the specification of cell fate during development.

Hedgehog (Hh) proteins play diverse organizing roles in development by regulating gene expression in responding cells. The Gli homolog Cubitus interruptus (Ci) is involved in controlling the transcription of Hh target genes. A repressor form of Ci arises in the absence of Hh signaling by proteolytic cleavage of intact Ci. We show that this cleavage is essential for limb patterning and is regulated by Hh in vivo. We provide evidence for the existence of a distinct activator form of Ci, which does not arise by mere prevention of Ci proteolysis, but rather depends on a separate regulatory step subject to Hh control. These different activities of Ci regulate overlapping but distinct subsets of Hh target genes. Thus, limb development is organized by the integration of different transcriptional outputs of Hh signaling.

The Drosophila Gli homolog Cubitus interruptus (Ci) 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, we dissect here the imaginal disc enhancer of the dpp gene, which responds to both activities of Ci. Within a minimal disc enhancer, we identify the DNA sequences that are necessary and sufficient for the control by Ci, show that the same sequences respond to the activator and repressor forms of Ci, and demonstrate that their activities can be replaced by a single synthetic Gli-binding site. We further show that 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.

Hedgehog controls the expression of key developmental genes through the conversion of the transcription factor Cubitus interruptus (Ci) into either an activator (Ci[act]) or a repressor (Ci[rep]) form. Proteolytic cleavage of full-length Ci is important for the generation of Ci[rep], but little is known about how Ci[act] arises in response to Hh. Here we examine Hh signal transduction components for their role in the conversion of full-length Ci into either Ci[act] or Ci[rep]. We report that Cos2, PKA and Fused are necessary for the generation of Ci[rep], whereas the inhibition of either Cos2 or PKA activity is a prerequisite for Ci[act] formation. Fused (Fu) kinase stimulates a constitutively active form of Ci in a Hh-dependent manner, suggesting that Fu enhances the activity rather than the formation of Ci[act]. Su(fu) reduces the nuclear accumulation of the constitutively active form of Ci, arguing that Su(fu) can function subsequent to Ci[act] formation. We propose that Hh induces target gene expression by a two-step mechanism in which Ci[act] is first formed and then accumulates in the nucleus via Fu-induced neutralization of Su(fu) activity.