Germ-line transformation via transposable elements is a powerful tool to study gene function in Drosophila melanogaster. However, some inherent characteristics of transposon-mediated transgenesis limit its use for transgene analysis. Here, we circumvent these limitations by optimizing a C31-based integration system. We generated a collection of lines with precisely mapped attP sites that allow the insertion of transgenes into many different predetermined intergenic locations throughout the fly genome. By using regulatory elements of the nanos and vasa genes, we established endogenous sources of the C31 integrase, eliminating the difficulties of coinjecting integrase mRNA and raising the transformation efficiency. Moreover, to discriminate between specific and rare nonspecific integration events, a white gene-based reconstitution system was generated that enables visual selection for precise attP targeting. Finally, we demonstrate that our chromosomal attP sites can be modified in situ, extending their scope while retaining their properties as landing sites. The efficiency, ease-of-use, and versatility obtained here with the C31-based integration system represents an important advance in transgenesis and opens up the possibility of systematic, highthroughput screening of large cDNA sets and regulatory elements.attP landing sites ͉ germ-line transformation ͉ site-specific integration A major goal in the present era of genomics is to identify and functionally characterize all genes relevant to a specific pathway or biological process. With its powerful repertoire of genetic tools, the multicellular model organism Drosophila melanogaster has played an eminent role in this endeavor (1). One method to identify relevant genes is to perform chemical mutagenesis screens of various kinds. Another very fruitful approach in Drosophila has been the use of P-element-mediated germ-line transformation (2, 3), especially when combined with tools such as the Gal4/UAS expression system (4) or when it is used for insertional mutagenesis (5, 6). One characteristic of P-elements is their random integration behavior. Although this ''randomness'' is advantageous for generating mutations and deletions, it is generally not ideal for transgene analysis. The random integration of P-elements necessitates considerable effort to map insertions. Genomic position effects complicate the analysis of transgenes and render precise structure/ function analyses nearly impossible. A further shortcoming of the P-element system is its relatively moderate transformation efficiency, a significant hurdle to any large-scale transgenesis effort.Strategies have been developed to circumvent the problem of randomness by targeted integration systems in Drosophila, which are generally based on the FLP and Cre recombinases (7-9, 32). Such techniques permit precise targeting to genomic landing sites but are still handicapped by transformation rates that are, at best, moderately higher than those achieved with the P-element system (8). Furthermore, especially for FL...

Switzerlandb-Catenin (Armadillo in Drosophila) is a multitasking and evolutionary conserved molecule that in metazoans exerts a crucial role in a multitude of developmental and homeostatic processes. More specifically, b-catenin is an integral structural component of cadherin-based adherens junctions, and the key nuclear effector of canonical Wnt signalling in the nucleus. Imbalance in the structural and signalling properties of b-catenin often results in disease and deregulated growth connected to cancer and metastasis. Intense research into the life of b-catenin has revealed a complex picture. Here, we try to capture the state of the art: we try to summarize and make some sense of the processes that regulate b-catenin, as well as the plethora of b-catenin binding partners. One focus will be the interaction of b-catenin with different transcription factors and the potential implications of these interactions for direct cross-talk between b-catenin and non-Wnt signalling pathways.

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.

Cell-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.

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.

Wnt/Wingless signaling controls many fundamental processes during animal development. Wnt transduction is mediated by the association of beta-catenin with nuclear TCF DNA binding factors. Here we report the identification of two segment polarity genes in Drosophila, legless (lgs), and pygopus (pygo), and we show that their products are required for Wnt signal transduction at the level of nuclear beta-catenin. Lgs encodes the homolog of human BCL9, and we provide genetic and molecular evidence that these proteins exert their function by physically linking Pygo to beta-catenin. Our results suggest that the recruitment of Pygo permits beta-catenin to transcriptionally activate Wnt target genes and raise the possibility that a deregulation of these events may play a causal role in the development of B cell malignancies.

The adult appendages of Drosophila are formed from imaginal discs, sheets of epithelial cells that proliferate during larval development and differentiate during metamorphosis. wingless (wg, DWnt-1) protein, a putative signaling molecule, is expressed only in prospective ventral cells in each of the leg discs. To test the role of wg, we have generated randomly positioned clones of cells that express wg protein constitutively. Clones that arise in the prospective ventral portions of the leg discs develop normally. In contrast, dorsally situated clones give rise to ventrolateral patterns and exert a ventralizing influence on neighboring wild-type tissue. We propose that wg protein organizes leg pattern along the dorsoventral axis by conferring ventral positional information within the disc. The adult appendages of Drosophila are formed from imaginal discs, sheets of epithelial cells that proliferate during larval development and differentiate during metamorphosis. wingless (wg, DWnt-1) protein, a putative signaling molecule, is expressed only in prospective ventral cells in each of the leg discs. To test the role of wg, we have generated randomly positioned clones of cells that express wg protein constitutively. Clones that arise in the prospective ventral portions of the leg discs develop normally. In contrast, dorsally situated clones give rise to ventrolateral patterns and exert a ventraliring influence on neighboring wild-type tissue. We propose that wg protein organizes leg pattern along the dorsoventral axis by conferring ventral positional information within the disc.