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The gastrointestinal tract has recently come to the forefront of multiple research fields. It is now recognized as a major source of signals modulating food intake, insulin secretion and energy balance. It is also a key player in immunity and, through its interaction with microbiota, can shape our physiology and behavior in complex and sometimes unexpected ways. The insect intestine had remained, by comparison, relatively unexplored until the identification of adult somatic stem cells in the Drosophila intestine over a decade ago. Since then, a growing scientific community has exploited the genetic amenability of this insect organ in powerful and creative ways. By doing so, we have shed light on a broad range of biological questions revolving around stem cells and their niches, interorgan signaling and immunity. Despite their relatively recent discovery, some of the mechanisms active in the intestine of flies have already been shown to be more widely applicable to other gastrointestinal systems, and may therefore become relevant in the context of human pathologies such as gastrointestinal cancers, aging, or obesity. This review summarizes our current knowledge of both the formation and function of the Drosophila melanogaster digestive tract, with a major focus on its main digestive/absorptive portion: the strikingly adaptable adult midgut.

Single nucleotide polymorphisms (SNPs) are useful markers for genetic mapping experiments in model organisms. Here we report the establishment of a high-density SNP map and high-throughput genotyping assays for Drosophila melanogaster . Our map comprises 27,367 SNPs in common laboratory Drosophila stocks. These SNPs were clustered within 2,238 amplifiable markers at an average density of 1 marker every 50.3 kb, or 6.3 genes. We have also constructed a set of 62 Drosophila stocks, each of which facilitates the generation of recombinants within a defined genetic interval of 1–2 Mb. For flexible, high-throughput SNP genotyping, we used fluorescent tag-array mini-sequencing (TAMS) assays. We designed and validated TAMS assays for 293 SNPs at an average resolution of 391.3 kb, and demonstrated the utility of these tools by rapidly mapping 14 mutations that disrupt embryonic muscle patterning. These resources enable high-resolution high-throughput genetic mapping in Drosophila .

The discovery of two genes encoded by prophage WO from Wolbachia that functionally recapitulate and enhance cytoplasmic incompatibility in arthropods is the first inroad in solving the genetic basis of reproductive parasitism. Manipulation of insect survival by Wolbachia bacteria Bacteria from the genus Wolbachia infect many arthropods, including the mosquitoes that are vectors for many viruses that infect humans. Wolbachia infection causes 'cytoplasmic incompatibility', which means that crosses between infected males and uninfected females lead to embryonic death, increasing the proportion of infected females in the population. The molecular basis for this effect has been unknown. Here, Seth Bordenstein and colleagues use comparative and transgenic approaches to identify two genes encoded by the prophage WO from Wolbachia that recapitulate cytoplasmic incompatibility. The discovery of these cytoplasmic incompatibility factors could lead to the genetic manipulation of WO-induced reproductive alterations, and may feed into efforts to control the transmission of arthropod-borne viruses to humans. The genus Wolbachia is an archetype of maternally inherited intracellular bacteria that infect the germline of numerous invertebrate species worldwide. They can selfishly alter arthropod sex ratios and reproductive strategies to increase the proportion of the infected matriline in the population. The most common reproductive manipulation is cytoplasmic incompatibility, which results in embryonic lethality in crosses between infected males and uninfected females. Females infected with the same Wolbachia strain rescue this lethality. Despite more than 40 years of research 1 and relevance to symbiont-induced speciation 2 , 3 , as well as control of arbovirus vectors 4 , 5 , 6 and agricultural pests 7 , the bacterial genes underlying cytoplasmic incompatibility remain unknown. Here we use comparative and transgenic approaches to demonstrate that two differentially transcribed, co-diverging genes in the eukaryotic association module of prophage WO 8 from Wolbachia strain w Mel recapitulate and enhance cytoplasmic incompatibility. Dual expression in transgenic, uninfected males of Drosophila melanogaster crossed to uninfected females causes embryonic lethality. Each gene additively augments embryonic lethality in crosses between infected males and uninfected females. Lethality associates with embryonic defects that parallel those of wild-type cytoplasmic incompatibility and is notably rescued by w Mel-infected embryos in all cases. The discovery of cytoplasmic incompatibility factor genes cifA and cifB pioneers genetic studies of prophage WO-induced reproductive manipulations and informs the continuing use of Wolbachia to control dengue and Zika virus transmission to humans.

We and others recently demonstrated that the readily programmable CRISPR/Cas9 system can be used to edit the Drosophila genome. However, most applications to date have relied on aberrant DNA repair to stochastically generate frameshifting indels and adoption has been limited by a lack of tools for efficient identification of targeted events. Here we report optimized tools and techniques for expanded application of the CRISPR/Cas9 system in Drosophila through homology-directed repair (HDR) with double-stranded DNA (dsDNA) donor templates that facilitate complex genome engineering through the precise incorporation of large DNA sequences, including screenable markers. Using these donors, we demonstrate the replacement of a gene with exogenous sequences and the generation of a conditional allele. To optimize efficiency and specificity, we generated transgenic flies that express Cas9 in the germline and directly compared HDR and off-target cleavage rates of different approaches for delivering CRISPR components. We also investigated HDR efficiency in a mutant background previously demonstrated to bias DNA repair toward HDR. Finally, we developed a web-based tool that identifies CRISPR target sites and evaluates their potential for off-target cleavage using empirically rooted rules. Overall, we have found that injection of a dsDNA donor and guide RNA-encoding plasmids into vasa-Cas9 flies yields the highest efficiency HDR and that target sites can be selected to avoid off-target mutations. Efficient and specific CRISPR/Cas9-mediated HDR opens the door to a broad array of complex genome modifications and greatly expands the utility of CRISPR technology for Drosophila research.

During organismal development, cells undergo complex changes in shape whose causal relationship to individual morphogenetic processes remains unclear. The modular nature of such processes suggests that it should be possible to isolate individual modules, determine the minimum set of requirements sufficient to drive tissue remodeling, and re-construct morphogenesis. Here we use optogenetics to reconstitute epithelial folding in embryonic Drosophila tissues that otherwise would not undergo invagination. We show that precise spatial and temporal activation of Rho signaling is sufficient to trigger apical constriction and tissue folding. Induced furrows can occur at any position along the dorsal–ventral or anterior–posterior embryo axis in response to the spatial pattern and level of optogenetic activation. Thus, epithelial folding is a direct function of the spatio-temporal organization and strength of Rho signaling that on its own is sufficient to drive tissue internalization independently of any pre-determined condition or differentiation program associated with endogenous invagination processes. Optogenetics is opening the possibility to not only perturb morphogenesis, but also to guide it. Here, the authors use this technique to reconstruct epithelial folding in Drosophila embryos and study the relationship between strength of Rho activation, apical constrictions, and tissue invagination.

By the onset of morphogenesis, Drosophila embryos consist of about 6000 cells that express distinct gene combinations. Here, we used single-cell sequencing of precisely staged embryos and devised DistMap, a computational mapping strategy to reconstruct the embryo and to predict spatial gene expression approaching single-cell resolution. We produced a virtual embryo with about 8000 expressed genes per cell. Our interactive Drosophila Virtual Expression eXplorer (DVEX) database generates three-dimensional virtual in situ hybridizations and computes gene expression gradients. We used DVEX to uncover patterned expression of transcription factors and long noncoding RNAs, as well as signaling pathway components. Spatial regulation of Hippo signaling during early embryogenesis suggests a mechanism for establishing asynchronous cell proliferation. Our approach is suitable to generate transcriptomic blueprints for other complex tissues.

The establishment of cell types during development requires precise interactions between genes and distal regulatory sequences. We have a limited understanding of how these interactions look in three dimensions, vary across cell types in complex tissue, and relate to transcription. Here we describe optical reconstruction of chromatin architecture (ORCA), a method that can trace the DNA path in single cells with nanoscale accuracy and genomic resolution reaching two kilobases. We used ORCA to study a Hox gene cluster in cryosectioned Drosophila embryos and labelled around 30 RNA species in parallel. We identified cell-type-specific physical borders between active and Polycomb-repressed DNA, and unexpected Polycomb-independent borders. Deletion of Polycomb-independent borders led to ectopic enhancer–promoter contacts, aberrant gene expression, and developmental defects. Together, these results illustrate an approach for high-resolution, single-cell DNA domain analysis in vivo, identify domain structures that change with cell identity, and show that border elements contribute to the formation of physical domains in Drosophila . Optical reconstruction of chromatin architecture and multiplex RNA labelling traces the DNA path in single cells and its relationship to transcription.

During gastrulation, physical forces reshape the simple embryonic tissue to form the complex body plans of multicellular organisms 1 . These forces often cause large-scale asymmetric movements of the embryonic tissue 2 , 3 . In many embryos, the gastrulating tissue is surrounded by a rigid protective shell 4 . Although it is well-recognized that gastrulation movements depend on forces that are generated by tissue-intrinsic contractility 5 , 6 , it is not known whether interactions between the tissue and the protective shell provide additional forces that affect gastrulation. Here we show that a particular part of the blastoderm tissue of the red flour beetle ( Tribolium castaneum ) tightly adheres in a temporally coordinated manner to the vitelline envelope that surrounds the embryo. This attachment generates an additional force that counteracts tissue-intrinsic contractile forces to create asymmetric tissue movements. This localized attachment depends on an αPS2 integrin (inflated), and the knockdown of this integrin leads to a gastrulation phenotype that is consistent with complete loss of attachment. Furthermore, analysis of another integrin (the αPS3 integrin, scab) in the fruit fly ( Drosophila melanogaster ) suggests that gastrulation in this organism also relies on adhesion between the blastoderm and the vitelline envelope. Our findings reveal a conserved mechanism through which the spatiotemporal pattern of tissue adhesion to the vitelline envelope provides controllable, counteracting forces that shape gastrulation movements in insects. In the red flour beetle ( Tribolium castaneum ) and fruit fly ( Drosophila melanogaster ), spatiotemporally coordinated integrin-dependent attachments between the blastoderm and vitelline envelope counteract tissue-intrinsic contractile forces to create asymmetric movements of embryonic tissue.

An improved assay for chromatin accessibility at single-cell resolution in Drosophila melanogaster embryos enables identification of developmental-stage- and cell-lineage-specific patterns of chromatin-level transcriptional regulation. Single-cell ATAC-seq in fly embryos Active gene regulatory elements shape the output of gene transcription and can be mapped across the genome by measuring chromatin accessibility. Eileen Furlong and colleagues apply a technique called ATAC sequencing to profile chromatin accessibility at a single-cell resolution during three stages of Drosophila embryogenesis. They map tissue-specific regulatory elements and show that the chromatin accessibility landscape is sufficient to infer individual cell types and developmental trajectories. A group of cells is found to use regulatory elements of both mesoderm and endoderm, which suggests the existence of a mesendoderm lineage in Drosophila . Understanding how gene regulatory networks control the progressive restriction of cell fates is a long-standing challenge. Recent advances in measuring gene expression in single cells are providing new insights into lineage commitment. However, the regulatory events underlying these changes remain unclear. Here we investigate the dynamics of chromatin regulatory landscapes during embryogenesis at single-cell resolution. Using single-cell combinatorial indexing assay for transposase accessible chromatin with sequencing (sci-ATAC-seq) 1 , we profiled chromatin accessibility in over 20,000 single nuclei from fixed Drosophila melanogaster embryos spanning three landmark embryonic stages: 2–4 h after egg laying (predominantly stage 5 blastoderm nuclei), when each embryo comprises around 6,000 multipotent cells; 6–8 h after egg laying (predominantly stage 10–11), to capture a midpoint in embryonic development when major lineages in the mesoderm and ectoderm are specified; and 10–12 h after egg laying (predominantly stage 13), when each of the embryo’s more than 20,000 cells are undergoing terminal differentiation. Our results show that there is spatial heterogeneity in the accessibility of the regulatory genome before gastrulation, a feature that aligns with future cell fate, and that nuclei can be temporally ordered along developmental trajectories. During mid-embryogenesis, tissue granularity emerges such that individual cell types can be inferred by their chromatin accessibility while maintaining a signature of their germ layer of origin. Analysis of the data reveals overlapping usage of regulatory elements between cells of the endoderm and non-myogenic mesoderm, suggesting a common developmental program that is reminiscent of the mesendoderm lineage in other species 2 , 3 , 4 . We identify 30,075 distal regulatory elements that exhibit tissue-specific accessibility. We validated the germ-layer specificity of a subset of these predicted enhancers in transgenic embryos, achieving an accuracy of 90%. Overall, our results demonstrate the power of shotgun single-cell profiling of embryos to resolve dynamic changes in the chromatin landscape during development, and to uncover the cis -regulatory programs of metazoan germ layers and cell types.

Sensory hairs on the back of a fruit fly are lined up in neat rows. The orderliness of this arrangement has encouraged models based on organized specification of the hairs. Corson et al. now show that development is both less precise and more effective than that. They used mathematical modeling to recapitulate genetic effects as the developing epidermis becomes organized into enough rows and single lines of hairs. Their work suggests that the sensory field develops through self-organizing patterning that can adjust to the size of the epidermis. Science , this issue p. eaai7407 Distributed and flexible patterning combines with cell-cell interactions to establish rows of sensory bristles on the fly thorax. The emergence of spatial patterns in developing multicellular organisms relies on positional cues and cell-cell communication. Drosophila sensory organs have informed a paradigm in which these operate in two distinct steps: Prepattern factors drive localized proneural activity, then Notch-mediated lateral inhibition singles out neural precursors. Here we show that self-organization through Notch signaling also establishes the proneural stripes that resolve into rows of sensory bristles on the fly thorax. Patterning, initiated by a gradient of Delta ligand expression, progresses through inhibitory signaling between and within stripes. Thus, Notch signaling can support self-organized tissue patterning as a prepattern is transduced by cell-cell interactions into a refined arrangement of cellular fates.