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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.

Anchor away is a sequestering method designed to acutely and timely abrogate the function of a protein of interest by anchoring to a cell compartment different from its target. This method induces the binding of the target protein to the anchor by either the addition of rapamycin to Drosophila food or cell media. Rapamycin mediates the formation of a ternary complex between the anchor, which is tagged with the FK506-binding protein (FKBP12), and the target protein fused with the FKB12 rapamycin-binding (FRB) domain of mammalian target of rapamycin (mTOR). The rapamycin-bound target protein stays sequestered away from its compartment, where it cannot perform its biological function.

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.

SummaryDrosophila melanogasterhas been an excellent model for innate immunity, but the role and regulation of adult blood cells and organismal immunity have remained incompletely understood. Here we address these questions in a comprehensive investigation of the blood cell system in adultDrosophila. As a central finding, we reveal the largest reservoir of blood cells (hemocytes) at the respiratory epithelia (tracheal air sacs) and fat body of the thorax and head. We show that most hemocytes of adultDrosophilaare phagocytic macrophages (plasmatocytes), derived by more than 60% from the embryonic lineage that parallels vertebrate tissue macrophages. Surprisingly, in contrast to hemocytes at the larval stage, we find no capacity of the adult blood cell system to expand. Instead, we demonstrate its central role in relaying an innate immune response to tissues surrounding the blood cell reservoir: Hemocytes, through Imd signaling and the Jak/Stat pathway ligand Upd3, act as sentinels of bacterial infection that induce expression of the antimicrobial peptide geneDrosocin in the respiratory epithelia and colocalizing domains of the fat body. We demonstrate that endogenousDrosocinexpression in these tissues promotes animal survival after bacterial infection. Our work identifies the first molecular step in a new relay of organismal immunity, establishing adultDrosophilaas model to dissect mechanisms of inter-organ immunity.

Drosophila melanogaster has been an excellent model for innate immunity, but the role and regulation of adult blood cells and organismal immunity have remained incompletely understood. Here we address these questions in a comprehensive investigation of the blood cell system in adult Drosophila. As a central finding, we reveal the largest reservoir of blood cells (hemocytes) at the respiratory epithelia (tracheal air sacs) and fat body of the thorax and head. We show that most hemocytes of adult Drosophila are phagocytic macrophages (plasmatocytes), derived by more than 60% from the embryonic lineage that parallels vertebrate tissue macrophages. Surprisingly, in contrast to hemocytes at the larval stage, we find no capacity of the adult blood cell system to expand. Instead, we demonstrate its central role in relaying an innate immune response to tissues surrounding the blood cell reservoir: Hemocytes, through Imd signaling and the Jak/Stat pathway ligand Upd3, act as sentinels of bacterial infection that induce expression of the antimicrobial peptide gene Drosocin in the respiratory epithelia and colocalizing domains of the fat body. We demonstrate that endogenous Drosocin expression in these tissues promotes animal survival after bacterial infection. Our work identifies the first molecular step in a new relay of organismal immunity, establishing adult Drosophila as model to dissect mechanisms of inter-organ immunity.

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. DOI: http://dx.doi.org/10.7554/eLife.22546.001, eLife digest From the wings of a butterfly to the fingers of a human hand, living tissues often have complex and intricate patterns. Developmental biologists have long been fascinated by the signals – called morphogens – that guide how these kinds of pattern develop. Morphogens are substances that are produced by groups of cells and spread to the rest of the tissue to form a gradient. Depending on where they sit along this gradient, cells in the tissue activate different sets of genes, and the resulting pattern of gene activity ultimately defines the position of the different parts of the tissue. Decades worth of studies into how limbs develop in animals from mice to fruit flies have revealed common principles of morphogen gradients that regulate the development of tissue patterns. Morphogens have been shown to help regulate the growth of tissues in a number of different animals as well. However, how the morphogens regulate tissue size and what role their gradients play in this process remain topics of intense debate in the field of developmental biology. In the developing wing of a fruit fly, a morphogen called Dpp is expressed in a thin stripe located in the centre and spreads to the rest of the tissue to form a gradient. Bosch, Ziukaite, Alexandre et al. have now characterised where and when the Dpp morphogen must be produced to regulate both the final size of the fly’s wing and the number of cells the wing eventually contains. The experiments involved preventing the production of Dpp in the developing wing in specific cells and at specific stages of development. This approach confirmed that Dpp must be produced in the central stripe for the wing to grow. Matsuda and Affolter and, independently, Barrio and Milán report the same findings in two related studies. Moreover, Bosch et al. and Barrio and Milán also conclude that the gradient of Dpp throughout the wing is not required for growth. Further work will be needed to explain how the Dpp signal regulates the growth of the wing. The answer to this question will contribute to a better understanding of the role of morphogens in regulating the size of human organs and how a failure to do so might cause developmental disorders. DOI: http://dx.doi.org/10.7554/eLife.22546.002

Summary The use of adult Drosophila melanogaster as a model for hematopoiesis or organismal immunity has been debated. Addressing this question, we identify an extensive reservoir of blood cells (hemocytes) at the respiratory epithelia (tracheal air sacs) of the thorax and head. Lineage tracing and functional analyses demonstrate that the majority of adult hemocytes are phagocytic macrophages (plasmatocytes) from the embryonic lineage that parallels vertebrate tissue macrophages. Surprisingly, we find no sign of adult hemocyte expansion. Instead, hemocytes play a role in relaying an innate immune response to the blood cell reservoir: through Imd signaling and the Jak/Stat pathway ligand Upd3, hemocytes act as sentinels of bacterial infection, inducing expression of the antimicrobial peptide Drosocin in respiratory epithelia and colocalizing fat body domains. Drosocin expression in turn promotes animal survival after infection. Our work identifies a multi-signal relay of organismal humoral immunity, establishing adult Drosophila as model for inter-organ immunity.