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Wnt proteins are members of a conserved family of secreted signaling ligands and play crucial roles during development and in tissue homeostasis. There is increasing evidence that aberrant Wnt production is an underlying cause of dysregulated Wnt signaling, however little is known about this process. One protein known to play a role in secretion is the transmembrane protein Wntless (Wls). However, the mechanism by which Wls promotes Wnt secretion is a riddle. It is not known which Wnt family members require Wls and what the structural requirements are that make some of them reliant on Wls for secretion. Here we present a systematic analysis of all known Drosophila Wnt family members with respect to their dependence on Wls function for secretion. We first show that the glycosylation status of Wg at conserved sites does not determine its dependence on Wls. Moreover, in apparent contrast to murine wls, Drosophila wls is not a target gene of canonical Wnt signaling. We then show that all Wnts, with the exception of WntD, require Wls for secretion. All Wnts, with the exception of WntD, also contain a conserved Serine residue (in Wg S239), which we show to be essential for their functional and physical interaction with Wls. Finally, all Wnts, with the exception of WntD, require the acyltransferase Porcupine for activity and for functionally interacting with Wls. Together, these findings indicate that Por-mediated lipidation of the S239-equivalent residue is essential for the interaction with, and secretion by, Wls.

Wnt proteins are lipid-modified glycoproteins that play a central role in development, adult tissue homeostasis and disease. Secretion of Wnt proteins is mediated by the Wnt-binding protein Wntless (Wls), which transports Wnt from the Golgi network to the cell surface for release. It has recently been shown that recycling of Wls through a retromer-dependent endosome-to-Golgi trafficking pathway is required for efficient Wnt secretion, but the mechanism of this retrograde transport pathway is poorly understood. Here, we report that Wls recycling is mediated through a retromer pathway that is independent of the retromer sorting nexins SNX1-SNX2 and SNX5-SNX6. We have found that the unrelated sorting nexin, SNX3, has an evolutionarily conserved function in Wls recycling and Wnt secretion and show that SNX3 interacts directly with the cargo-selective subcomplex of the retromer to sort Wls into a morphologically distinct retrieval pathway. These results demonstrate that SNX3 is part of an alternative retromer pathway that functionally separates the retrograde transport of Wls from other retromer cargo. [KEYWORDS: Animals, Animals, Genetically Modified, Biological Transport, Active, Caenorhabditis elegans/genetics/growth & development/metabolism, Drosophila/genetics/growth & development/metabolism, Endosomes/metabolism, HeLa Cells, Humans, Intracellular Signaling Peptides and Proteins/ metabolism, Models, Biological, RNA Interference, Signal Transduction, Sorting Nexins/antagonists & inhibitors/genetics/ metabolism, Wnt Proteins/ metabolism, trans-Golgi Network/metabolism], Wnt proteins are lipid-modified glycoproteins that play a central role in development, adult tissue homeostasis and disease. Secretion of Wnt proteins is mediated by the Wnt-binding protein Wntless (Wls), which transports Wnt from the Golgi network to the cell surface for release. It has recently been shown that recycling of Wls through a retromer-dependent endosome-to-Golgi trafficking pathway is required for efficient Wnt secretion, but the mechanism of this retrograde transport pathway is poorly understood. Here, we report that Wls recycling is mediated through a retromer pathway that is independent of the retromer sorting nexins SNX1-SNX2 and SNX5-SNX6. We have found that the unrelated sorting nexin, SNX3, has an evolutionarily conserved function in Wls recycling and Wnt secretion and show that SNX3 interacts directly with the cargo-selective subcomplex of the retromer to sort Wls into a morphologically distinct retrieval pathway. These results demonstrate that SNX3 is part of an alternative retromer pathway that functionally separates the retrograde transport of Wls from other retromer cargo. [KEYWORDS: Animals, Animals, Genetically Modified, Biological Transport, Active, Caenorhabditis elegans/genetics/growth & development/metabolism, Drosophila/genetics/growth & development/metabolism, Endosomes/metabolism, HeLa Cells, Humans, Intracellular Signaling Peptides and Proteins/ metabolism, Models, Biological, RNA Interference, Signal Transduction, Sorting Nexins/antagonists & inhibitors/genetics/ metabolism, Wnt Proteins/ metabolism, trans-Golgi Network/metabolism]

Extended abstract of a paper presented at Microscopy and Microanalysis 2011 in Nashville, Tennessee, USA, August 7-August 11, 2011

Non-intermingling, adjacent populations of cells define compartment boundaries; such boundaries are often essential for the positioning and the maintenance of tissue-organizers during growth. In the developing wing primordium of Drosophila melanogaster, signaling by the secreted protein Hedgehog (Hh) is required for compartment boundary maintenance. However, the precise mechanism of Hh input remains poorly understood. Here, we combine experimental observations of perturbed Hh signaling with computer simulations of cellular behavior, and connect physical properties of cells to their Hh signaling status. We find that experimental disruption of Hh signaling has observable effects on cell sorting surprisingly far from the compartment boundary, which is in contrast to a previous model that confines Hh influence to the compartment boundary itself. We have recapitulated our experimental observations by simulations of Hh diffusion and transduction coupled to mechanical tension along cell-to-cell contact surfaces. Intriguingly, the best results were obtained under the assumption that Hh signaling cannot alter the overall tension force of the cell, but will merely re-distribute it locally inside the cell, relative to the signaling status of neighboring cells. Our results suggest a scenario in which homotypic interactions of a putative Hh target molecule at the cell surface are converted into a mechanical force. Such a scenario could explain why the mechanical output of Hh signaling appears to be confined to the compartment boundary, despite the longer range of the Hh molecule itself. Our study is the first to couple a cellular vertex model describing mechanical properties of cells in a growing tissue, to an explicit model of an entire signaling pathway, including a freely diffusible component. We discuss potential applications and challenges of such an approach.

Wnt proteins are lipid-modified glycoproteins that have important roles in development, adult tissue homeostasis and disease. Secretion of Wnt proteins from producing cells is mediated by the Wnt-binding protein MIG-14/Wls, which binds Wnt in the Golgi network and transports it to the cell surface for release. It has recently been shown that recycling of MIG-14/Wls from the plasma membrane to the trans-Golgi network is required for efficient Wnt secretion, but the mechanism of this retrograde transport pathway is still poorly understood. In this study, we report the identification of MTM-6 and MTM-9 as novel regulators of MIG-14/Wls trafficking in Caenorhabditis elegans. MTM-6 and MTM-9 are myotubularin lipid phosphatases that function as a complex to dephosphorylate phosphatidylinositol-3-phosphate, a central regulator of endosomal trafficking. We show that mutation of mtm-6 or mtm-9 leads to defects in several Wnt-dependent processes and demonstrate that MTM-6 is required in Wnt-producing cells as part of the MIG-14/Wls-recycling pathway. This function is evolutionarily conserved, as the MTM-6 orthologue DMtm6 is required for Wls stability and Wg secretion in Drosophila. We conclude that regulation of endosomal trafficking by the MTM-6/MTM-9 myotubularin complex is required for the retromer-dependent recycling of MIG-14/Wls and Wnt secretion., Wnt proteins are lipid-modified glycoproteins that have important roles in development, adult tissue homeostasis and disease. Secretion of Wnt proteins from producing cells is mediated by the Wnt-binding protein MIG-14/Wls, which binds Wnt in the Golgi network and transports it to the cell surface for release. It has recently been shown that recycling of MIG-14/Wls from the plasma membrane to the trans-Golgi network is required for efficient Wnt secretion, but the mechanism of this retrograde transport pathway is still poorly understood. In this study, we report the identification of MTM-6 and MTM-9 as novel regulators of MIG-14/Wls trafficking in Caenorhabditis elegans. MTM-6 and MTM-9 are myotubularin lipid phosphatases that function as a complex to dephosphorylate phosphatidylinositol-3-phosphate, a central regulator of endosomal trafficking. We show that mutation of mtm-6 or mtm-9 leads to defects in several Wnt-dependent processes and demonstrate that MTM-6 is required in Wnt-producing cells as part of the MIG-14/Wls-recycling pathway. This function is evolutionarily conserved, as the MTM-6 orthologue DMtm6 is required for Wls stability and Wg secretion in Drosophila. We conclude that regulation of endosomal trafficking by the MTM-6/MTM-9 myotubularin complex is required for the retromer-dependent recycling of MIG-14/Wls and Wnt secretion.

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.

Di- and trimethylation of histone H3 lysine 4 (H3K4me2 and H3K4me3) are hallmarks of chromatin at active genes. The major fraction of K4-methylated histone H3 is the variant H3 (termed H3.3 in Drosophila), which replaces canonical H3 (H3.2) in transcribed genes. 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.

BACKGROUND: The JNK pathway is a mitogen-activated protein (MAP) kinase pathway involved in the regulation of numerous physiological processes during development and in response to environmental stress. JNK activity is controlled by two MAPK kinases (MAPKK), Mkk4 and Mkk7. Mkk7 plays a prominent role upon Tumor Necrosis Factor (TNF) stimulation. Eiger, the unique TNF-superfamily ligand in Drosophila, potently activates JNK signaling through the activation of the MAPKKK Tak1. METHODOLOGY/PRINCIPAL FINDINGS: In a dominant suppressor screen for new components of the Eiger/JNK-pathway in Drosophila, we have identified an allelic series of the Mkk4 gene. Our genetic and biochemical results demonstrate that Mkk4 is dispensable for normal development and host resistance to systemic bacterial infection but plays a non-redundant role as a MAPKK acting in parallel to Hemipterous/Mkk7 in dTAK1-mediated JNK activation upon Eiger and Imd pathway activation. CONCLUSIONS/SIGNIFICANCE: In contrast to mammals, it seems that in Drosophila both MAPKKs, Hep/Mkk7 and Mkk4, are required to induce JNK upon TNF or pro-inflammatory stimulation.

Protein modifications play a major role for most biological processes in living organisms. Amino-terminal acetylation of proteins is a common modification found throughout the tree of life: the N-terminus of a nascent polypeptide chain becomes co-translationally acetylated, often after the removal of the initiating methionine residue. While the enzymes and protein complexes involved in these processes have been extensively studied, only little is known about the biological function of such N-terminal modification events. To identify common principles of N-terminal acetylation, we analyzed the amino-terminal peptides from proteins extracted from Drosophila Kc167 cells. We detected more than 1,200 mature protein N-termini and could show that N-terminal acetylation occurs in insects with a similar frequency as in humans. As the sole true determinant for N-terminal acetylation we could extract the (X)PX rule that indicates the prevention of acetylation under all circumstances. We could show that this rule can be used to genetically engineer a protein to study the biological relevance of the presence or absence of an acetyl group, thereby generating a generic assay to probe the functional importance of N-terminal acetylation. We applied the assay by expressing mutated proteins as transgenes in cell lines and in flies. Here, we present a straightforward strategy to systematically study the functional relevance of N-terminal acetylations in cells and whole organisms. Since the (X)PX rule seems to be of general validity in lower as well as higher eukaryotes, we propose that it can be used to study the function of N-terminal acetylation in all species.

The site-specific recombinase FLP is used in Drosophila to precisely manipulate the genome, in particular, to eliminate gene function by mitotic recombination and to activate transgenes in discrete populations of cells. These approaches are already part of the standard tool kit for studying gene function. The number of applications for the FLP recombinase has increased over the years and further members of the large family of site-specific recombinases are being added to the arsenal of fly geneticists, most recently, the phiC31 integrase. This chapter will introduce these recombinases and describe how such instruments are utilized to accurately manipulate the Drosophila genome.

The lipid-modified morphogens Wnt and Hedgehog diffuse poorly in isolation yet can spread over long distances in vivo, predicting existence of two distinct forms of these morphogens. The first is poorly mobile and activates short-range target genes. The second is specifically packed for efficient spreading to induce long-range targets. Subcellular mechanisms involved in the discriminative secretion of these two forms remain elusive. Wnt and Hedgehog can associate with membrane microdomains, but the function of this association was unknown. Here we show that a major protein component of membrane microdomains, reggie-1/flotillin-2, plays important roles in secretion and spreading of Wnt and Hedgehog in Drosophila. Reggie-1 loss-of-function results in reduced spreading of the morphogens, while its overexpression stimulates secretion of Wnt and Hedgehog and expands their diffusion. The resulting changes in the morphogen gradients differently affect the short- and long-range targets. In its action reggie-1 appears specific for Wnt and Hedgehog. These data suggest that reggie-1 is an important component of the Wnt and Hedgehog secretion pathway dedicated to formation of the mobile pool of these morphogens.

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.

Small insertions or deletions (InDels) constitute a ubiquituous class of sequence polymorphisms found in eukaryotic genomes. Here, we present an automated high-throughput genotyping method that relies on the detection of fragment-length polymorphisms (FLPs) caused by InDels. The protocol utilizes standard sequencers and genotyping software. We have established genome-wide FLP maps for both Caenorhabditis elegans and Drosophila melanogaster that facilitate genetic mapping with a minimum of manual input and at comparatively low cost.

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 over-proliferate. 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 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.

We report the use of the cross-linking drug hexamethylphosphoramide (HMPA), which introduces small deletions, as a mutagen suitable for reverse genetics in the model organism Drosophila melanogaster. A compatible mutation-detection method based on resolution of PCR fragment-length polymorphisms on standard DNA sequencers is implemented. As the spectrum of HMPA-induced mutations is similar in a variety of organisms, it should be possible to transfer this mutagenesis and detection procedure to other model systems.

The spatial and temporal control of gene expression during the development of multicellular organisms is regulated to a large degree by cell-cell signaling. We have uncovered a simple mechanism through which Dpp, a TGFbeta/BMP superfamily member in Drosophila, represses many key developmental genes in different tissues. A short DNA sequence, a Dpp-dependent silencer element, is sufficient to confer repression of gene transcription upon Dpp receptor activation and nuclear translocation of Mad and Medea. Transcriptional repression does not require the cooperative action of cell type-specific transcription factors but relies solely on the capacity of the silencer element to interact with Mad and Medea and to subsequently recruit the zinc finger-containing repressor protein Schnurri. Our findings demonstrate how the Dpp pathway can repress key targets in a simple and tissue-unrestricted manner in vivo and hence provide a paradigm for the inherent capacity of a signaling system to repress transcription upon pathway activation.

The Wingless (Wg) protein is a secreted glycoprotein involved in intercellular signaling. On activation of the Wg signaling pathway, Armadillo is stabilized, causing target genes to be activated by the transcription factor Pangolin (Pan). This study investigated the roles of Pan in the developing wing of Drosophila by clonal analysis. Three different aspects of wing development were examined: cell proliferation, wing margin specification, and wg self-refinement. Our results indicate that Pan function is critically required for all three of these processes. Consequently, lack of pan causes a severe reduction in the activity of the Wg target genes Distalless and vestigial within their normal domain of expression. Loss of pan function does not, however, lead to a derepression of these genes outside this domain. Thus, although Pan is positively required for the induction of Wg targets in the wing imaginal disk, it does not appear to play a default repressor function in the absence of Wg input. In contrast, lack of zygotic pan function causes a milder phenotype than that caused by the lack of wg function in the embryo. We show that this difference cannot be attributed to maternally provided pan product, indicating that a Pan repressor function usually prevents the expression of embryonic Wg targets. Together, our results suggest that for embryonic patterning the activator as well as repressor forms of Pan play important roles, while for wing development Pan operates primarily in the activator mode.

Morphogen gradients control body pattern by differentially regulating cellular behavior. Here, we analyze the molecular events underlying the primary response to the Dpp/BMP morphogen in Drosophila. Throughout development, Dpp transduction causes the graded transcriptional downregulation of the brinker (brk) gene. We first provide significance for the brk expression gradient by showing that different Brk levels repress distinct combinations of wing genes expressed at different distances from Dpp-secreting cells. We then dissect the brk regulatory region and identify two separable elements with opposite properties, a constitutive enhancer and a Dpp morphogen-regulated silencer. Furthermore, we present genetic and biochemical evidence that the brk silencer serves as a direct target for a protein complex consisting of the Smad homologs Mad/Medea and the zinc finger protein Schnurri. Together, our results provide the molecular framework for a mechanism by which the extracellular Dpp/BMP morphogen establishes a finely tuned, graded read-out of transcriptional repression.

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.

The dispatched (disp) gene is required for long-range Hedgehog (Hh) signaling in Drosophila. Here, we demonstrate that one of two murine homologs, mDispA, can rescue disp function in Drosophila and is essential for all Hh patterning activities examined in the early mouse embryo. Embryonic fibroblasts lacking mDispA respond normally to exogenously provided Sonic hedgehog (Shh) signal, but are impaired in stimulation of other responding cells when expressing Shh. We have developed a biochemical assay that directly measures the activity of Disp proteins in release of soluble Hh proteins. This activity is disrupted by alteration of residues functionally conserved in Patched and in a related family of bacterial transmembrane transporters, thus suggesting similar mechanisms of action for all of these proteins.

A collection of 1609 recessive P-lethal mutations on the third chromosome was tested in germline clones for effects on egg differentiation and embryonic development. In 164 lines, normal differentiation of the egg chamber is prevented and in 841 lines, embryos develop abnormally. This latter group of maternal-effect mutations was subdivided into 23 classes based on the cuticular phenotypes. Our collection comprises new alleles of previously characterized genes (e.g. kayak, punt, string, tramtrack). For some of the genes identified in this screen, a maternal contribution to embryonic development has not been described previously (e.g. extramacrochaete, Trithorax-like, single minded, couch potato, canoe). The genes classified in our study with a dual function during oogenesis and embryogenesis not only substantially extends the existing collection of maternal-effect genes but will also aid further understanding of how patterning of the Drosophila embryo is controlled by the maternal genome.

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.

decapentaplegic (dpp) encodes a Drosophila transforming growth factor-beta homologue that functions as a morphogen in the developing embryo and in adult appendage formation. In the wing imaginal disc, a Dpp gradient governs patterning along the anteroposterior axis by inducing regional expression of diverse genes in a concentration-dependent manner. Recent studies show that responses to graded Dpp activity also require an input from a complementary and opposing gradient of Brinker (Brk), a transcriptional repressor protein encoded by a Dpp target gene. Here we show that Brk harbours a functional and transferable repression domain, through which it recruits the corepressors Groucho and CtBP. By analysing transcriptional outcomes arising from the genetic removal of these corepressors, and by ectopically expressing Brk variants in the embryo, we demonstrate that these corepressors are alternatively used by Brk for repressing some Dpp-responsive genes, whereas for repressing other distinct target genes they are not required. Our results show that Brk utilizes multiple means to repress its endogenous target genes, allowing repression of a multitude of complex Dpp target promoters.

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.

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.

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.

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.

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.

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.

The mammalian growth factor TGFbeta negatively regulates cell proliferation in various systems. Here we provide evidence that another TGFbeta superfamily member, Drosophila Decapentaplegic (Dpp), stimulates cell proliferation. In the developing wing blade, somatic clones lacking the Dpp receptors Punt or Thick veins (Tkv), or lacking Schnurri, a transcription factor involved in Dpp signal interpretation, fail to grow when induced early in larval development. Furthermore the spatial requirement for these signaling components indicates that Dpp has to travel several cell diameters from its source in order to reach all cells that require its signal. The requirement for Tkv also depends on the distance of cells from the source of the Dpp signal. We propose that Dpp can act at a distance to positively control cell proliferation.

During development of the Drosophila wing, the decapentaplegic (dpp) gene is expressed in a stripe of cells along the anteroposterior compartment boundary and gives rise to a secreted protein that exerts a long-range organizing influence on both compartments. Using clones of cells that express DPP, or in which DPP receptor activity has been constitutively activated or abolished, we show that DPP acts directly and at long range on responding cells, rather than by proxy through the short-range induction of other signaling molecules. Further, we show that two genes, optomotor-blind and spalt are transcriptionally activated at different distances from DPP-secreting cells and provide evidence that these genes respond to different threshold concentrations of DPP protein. We propose that DPP acts as a gradient morphogen during wing development.

Wingless (Wg), a founding member of the Wingless/Int-1 (Wnt) family of secreted proteins, acts as a short-range inducer and as a long-range organizer during Drosophila development. Here, we determine the consequences of ectopically expressing (i) a wild-type form of Wg, (ii) a membrane-tethered form of Wg, and (iii) a constitutively active form of the cytosolic protein Armadillo (Arm), which normally acts to transduce Wg, and we compare them with the effects of removing endogenous Wg or Arm activity. Our results indicate that wild-type Wg acts at long range, up-regulating the transcription of particular target genes as a function of concentration and distance from secreting cells. In contrast, tethered Wg and Arm have only short-range or autonomous effects, respectively, on the transcription of these genes. We interpret these findings as evidence that Wg can act directly and at long range as a gradient morphogen during normal development.