Flamingo participates in multiple models of cell competition.

The growth and survival of cells with different fitness, such as those with a proliferative advantage or a deleterious mutation, is controlled through cell competition. During development, cell competition enables healthy cells to eliminate less fit cells that could jeopardize tissue integrity, and facilitates the elimination of pre-malignant cells by healthy cells as a surveillance mechanism to prevent oncogenesis. Malignant cells also benefit from cell competition to promote their expansion. Despite its ubiquitous presence, the mechanisms governing cell competition, particularly those common to developmental competition and tumorigenesis, are poorly understood. Here, we show that in Drosophila, the planar cell polarity (PCP) protein Flamingo (Fmi) is required by winners to maintain their status during cell competition in malignant tumors to overtake healthy tissue, in early pre-malignant cells when they overproliferate among wildtype cells, in healthy cells when they later eliminate pre-malignant cells, and by supercompetitors as they compete to occupy excessive territory within wildtype tissues. "Would-be" winners that lack Fmi are unable to over-proliferate, and instead become losers. We demonstrate that the role of Fmi in cell competition is independent of PCP, and that it uses a distinct mechanism that may more closely resemble one used in other less well-defined functions of Fmi.

Automated counting of Drosophila imaginal disc cell nuclei.

Automated image quantification workflows have dramatically improved over the past decade, enriching image analysis and enhancing the ability to achieve statistical power. These analyses have proved especially useful for studies in organisms such as Drosophila melanogaster, where it is relatively simple to obtain high sample numbers for downstream analyses. However, the developing wing, an intensively utilized structure in developmental biology, has eluded efficient cell counting workflows due to its highly dense cellular population. Here, we present efficient automated cell counting workflows capable of quantifying cells in the developing wing. Our workflows can count the total number of cells or count cells in clones labeled with a fluorescent nuclear marker in imaginal discs. Moreover, by training a machine-learning algorithm we have developed a workflow capable of segmenting and counting twin-spot labeled nuclei, a challenging problem requiring distinguishing heterozygous and homozygous cells in a background of regionally varying intensity. Our workflows could potentially be applied to any tissue with high cellular density, as they are structure-agnostic, and only require a nuclear label to segment and count cells.

Protein phosphatase 1 regulates core PCP signaling.

PCP signaling polarizes epithelial cells within the plane of an epithelium. Core PCP signaling components adopt asymmetric subcellular localizations within cells to both polarize and coordinate polarity between cells. Achieving subcellular asymmetry requires additional effectors, including some mediating post-translational modifications of core components. Identification of such proteins is challenging due to pleiotropy. We used mass spectrometry-based proximity labeling proteomics to identify such regulators in the Drosophila wing. We identified the catalytic subunit of Protein Phosphatase1, Pp1-87B, and show that it regulates core protein polarization. Pp1-87B interacts with the core protein Van Gogh and at least one Serine/Threonine kinase, Dco/CKIε, that is known to regulate PCP. Pp1-87B modulates Van Gogh subcellular localization and directs its dephosphorylation in vivo. PNUTS, a Pp1 regulatory subunit, also modulates PCP. While the direct substrate(s) of Pp1-87B in control of PCP is not known, our data support the model that cycling between phosphorylated and unphosphorylated forms of one or more core PCP components may regulate acquisition of asymmetry. Finally, our screen serves as a resource for identifying additional regulators of PCP signaling.

Anchor Away - A Fast, Reliable and Reversible Technique To Inhibit Proteins in Drosophila melanogaster.

Several techniques have been developed to study specific gene function in loss-of-function situations. In Drosophila melanogaster, RNAi and the generation of mutant clones are widely used. However, both techniques have the limitation that there is a significant time lag before gene function is abolished. Given the relatively rapid development of Drosophila, such perdurance is a serious impediment to study gene function. Here we describe the adaptation of the anchor-away technique for use in Drosophila Anchor-away was originally developed in yeast to quickly and efficiently abrogate the function of nuclear proteins by sequestering - anchoring - them away in a different cellular compartment. The required components are present in the cells, and the system is triggered by the addition of rapamycin, resulting in a rapid generation of a loss-of-function situation. We provide here proof of principle for the system by producing loss-of-function situations for two nuclear proteins - Pygopus and Brinker. The system allows to study the requirement of any protein during any time window, and at the same time circumvents difficulties, such as off-target effects or variable phenotypes, which are inherent in other techniques, for example RNAi.

Dpp controls growth and patterning in Drosophila wing precursors through distinct modes of action.

Dpp, a member of the BMP family, is a morphogen that specifies positional information in Drosophila wing precursors. In this tissue, Dpp expressed along the anterior-posterior boundary forms a concentration gradient that controls the expression domains of target genes, which in turn specify the position of wing veins. Dpp also promotes growth in this tissue. The relationship between the spatio-temporal profile of Dpp signalling and growth has been the subject of debate, which has intensified recently with the suggestion that the stripe of Dpp is dispensable for growth. With two independent conditional alleles of dpp, we find that the stripe of Dpp is essential for wing growth. We then show that this requirement, but not patterning, can be fulfilled by uniform, low level, Dpp expression. Thus, the stripe of Dpp ensures that signalling remains above a pro-growth threshold, while at the same time generating a gradient that patterns cell fates.