In most cancers, transformation begins in a single cell in an epithelial cell sheet. However, it is not known what happens at the interface between non-transformed (normal) and transformed cells once the initial transformation has occurred. Using Madin-Darby canine kidney (MDCK) epithelial cells that express constitutively active, oncogenic Ras (Ras(V12)) in a tetracycline-inducible system, we investigated the cellular processes arising at the interface between normal and transformed cells. We show that two independent phenomena occur in a non-cell-autonomous manner: when surrounded by normal cells, Ras(V12) cells are either apically extruded from the monolayer, or form dynamic basal protrusions and invade the basal matrix. Neither apical extrusion nor basal protrusion formation is observed when Ras(V12) cells are surrounded by other Ras(V12) cells. We show that Cdc42 and ROCK (also known as Rho kinase) have vital roles in these processes. We also demonstrate that E-cadherin knockdown in normal cells surrounding Ras(V12) cells reduces the frequency of apical extrusion, while promoting basal protrusion formation and invasion. These results indicate that Ras(V12)-transformed cells are able to recognize differences between normal and transformed cells, and consequently leave epithelial sheets either apically or basally, in a cell-context-dependent manner.

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Wnts are evolutionarily conserved secreted signalling proteins that, in various developmental contexts, spread from their site of synthesis to form a gradient and activate target-gene expression at a distance. However, the requirement for Wnts to spread has never been directly tested. Here we used genome engineering to replace the endogenous wingless gene, which encodes the main Drosophila Wnt, with one that expresses a membrane-tethered form of the protein. Surprisingly, the resulting flies were viable and produced normally patterned appendages of nearly the right size, albeit with a delay. We show that, in the prospective wing, prolonged wingless transcription followed by memory of earlier signalling allows persistent expression of relevant target genes. We suggest therefore that the spread of Wingless is dispensable for patterning and growth even though it probably contributes to increasing cell proliferation.

The glycolipoproteins of the Wnt family raise interesting trafficking issues, especially with respect to spreading within tissues. Recently, the retromer complex has been suggested to participate in packaging Wnts into long-range transport vehicles. Our analysis of a Drosophila mutant in Vps35 show that, instead, the retromer complex is required for efficient progression of Wingless (a Drosophila Wnt) through the secretory pathway. Indeed expression of senseless, a short-range target gene, is lost in Vps35-deficient imaginal discs. In contrast, Vps35 is not required for Hedgehog secretion, suggesting specificity. Overexpression of Wntless, a transmembrane protein known to be specifically required for Wingless secretion overcomes the secretion block of Vps35-mutant cells. Furthermore, biochemical evidence confirms that Wntless engages with the retromer complex. We propose that Wntless accompanies Wingless to the plasma membrane where the two proteins dissociate. Following dissociation from Wingless, Wntless is internalized and returns to the Golgi apparatus in a retromer-dependent manner. Without the retromer-dependent recycling route, Wingless secretion is impaired and, as electron microscopy suggests, Wntless is diverted to a degradative compartment.

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Gene targeting by ‘ends-out’ homologous recombination enables the deletion of genomic sequences and concurrent introduction of exogenous DNA with base-pair precision without sequence constraint. In Drosophila, this powerful technique has remained laborious and hence seldom implemented. We describe a targeting vector and protocols that achieve this at high frequency and with very few false positives in Drosophila, either with a two-generation crossing scheme or by direct injection in embryos. The frequency of injection-mediated gene targeting can be further increased with CRISPR-induced double-strand breaks within the region to be deleted, thus making homologous recombination almost as easy as conventional transgenesis. Our targeting vector replaces genomic sequences with a multifunctional fragment comprising an easy-to-select genetic marker, a fluorescent reporter, as well as an attP site, which acts as a landing platform for reintegration vectors. These vectors allow the insertion of a variety of transcription reporters or cDNAs to express tagged or mutant isoforms at endogenous levels. In addition, they pave the way for difficult experiments such as tissue-specific allele switching and functional analysis in post-mitotic or polyploid cells. Therefore, our method retains the advantages of homologous recombination while capitalising on the mutagenic power of CRISPR.

Embryos have evolved various strategies to confine the action of secreted signals. Using an HRP-Wingless fusion protein to track the fate of endocytosed Wingless, we show that degradation by targeting to lysosomes is one such strategy. Wingless protein is specifically degraded at the posterior of each stripe of wingless transcription, even under conditions of overexpression. If lysosomal degradation is compromised genetically or chemically, excess Wingless accumulates and ectopic signaling ensues. In the wild-type, Wingless degradation is slower at the anterior than at the posterior. This follows in part from the segmental activation of signaling by the epidermal growth factor receptor, which accelerates Wingless degradation at the posterior, thus leading to asymmetrical Wingless signaling along the anterior-posterior axis.

Limb development in Drosophila depends on subdivision of the limb primordia into functional units called compartments. Cell interactions across compartment boundaries establish pattern-organizing centres that control growth and specify cell fates along the anteroposterior (AP) and dorsoventral (DV) axes of the limbs. AP subdivision of the disc primordia is inherited from the embryonic ectoderm. DV subdivision of the wing disc occurs during the second larval instar through localized expression of the apterous protein (Apterous) in dorsal cells. A third major subdivision of the wing disc into wing and body-wall compartments also occurs in the second instar. Here we show that specification of the wing primordium in early second instar depends on activity of the AP patterning system but not the DV system. These results define two distinct roles for the wingless gene: a primary role in specifying the wing primordium, and a subsequent role mediating the patterning activities of the DV compartment boundary.