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Argonaute proteins of the PIWI clade complexed with PIWI-interacting RNAs (piRNAs) protect the animal germline genome by silencing transposable elements. One of the leading experimental systems for studying piRNA biology is the Drosophila melanogaster ovary. In addition to classical mutagenesis, transgenic RNA interference (RNAi), which enables tissue-specific silencing of gene expression, plays a central role in piRNA research. Here, we establish a versatile toolkit focused on piRNA biology that combines germline transgenic RNAi, GFP marker lines for key proteins of the piRNA pathway, and reporter transgenes to establish genetic hierarchies. We compare constitutive, pan-germline RNAi with an equally potent transgenic RNAi system that is activated only after germ cell cyst formation. Stage-specific RNAi allows us to investigate the role of genes essential for germline cell survival, for example, nuclear RNA export or the SUMOylation pathway, in piRNA-dependent and independent transposon silencing. Our work forms the basis for an expandable genetic toolkit provided by the Vienna Drosophila Resource Center., In eukaryotes, small RNA silencing pathways play fundamental roles in transposon control, which is of critical importance for genome stability. Drosophila is a leading study system for small RNA-guided transposon defense. Here, ElMaghraby, Tirian et al. present a set of genetic tools centered on transgenic RNA interference that will allow the dissection of the Piwi/piRNA pathway in the developing Drosophila ovary. This toolkit is particularly suited to study the intersection of piRNA biology and essential cellular processes.

It was long thought that solely three different transposable elements - the I-element, the P-element and hobo - invaded naturalD. melanogasterpopulations within the last century. By sequencing the ‘living fossils’ ofDrosophilaresearch, i.e.D. melanogasterstrains sampled from natural populations at different time points, we show that a fourth TE, Tirant, invadedD. melanogasterpopulations during the past century. Tirant likely spread inD. melanogasterpopulations around 1938, followed by the I-element, hobo, and, lastly, the P-element. In addition to the recent insertions of the canonical Tirant,D. melanogasterstrains harbour degraded Tirant sequences in the heterochromatin which are likely due to an ancient invasion, possibly predating the split ofD. melanogasterandD. simulans. In contrast to the I-element, P-element and hobo, we did not find that Tirant induces any hybrid dysgenesis symptoms. This absence of apparent phenotypic effects may explain the late discovery of the Tirant invasion. Recent Tirant insertions were found in all investigated natural populations. Populations from Tasmania carry distinct Tirant sequences, likely due to a founder effect. By investigating the TE composition of natural populations and strains sampled at different time points, insertion site polymorphisms, piRNAs and phenotypic effects, we provide a comprehensive study of a natural TE invasion.

Summary In animal gonads, PIWI proteins and their bound 23–30 nt piRNAs guard genome integrity by the sequence specific silencing of transposons. Two branches of piRNA biogenesis, namely primary processing and ping-pong amplification, have been proposed. Despite an overall conceptual understanding of piRNA biogenesis, identity and/or function of the involved players are largely unknown. Here, we demonstrate an essential role for the female sterility gene shutdown in piRNA biology. Shutdown, an evolutionarily conserved cochaperone collaborates with Hsp90 during piRNA biogenesis, potentially at the loading step of RNAs into PIWI proteins. We demonstrate that Shutdown is essential for both primary and secondary piRNA populations in Drosophila. An extension of our study to previously described piRNA pathway members revealed three distinct groups of biogenesis factors. Together with data on how PIWI proteins are wired into primary and secondary processing, we propose a unified model for piRNA biogenesis., Highlights ► The cochaperone Shutdown is an essential piRNA biogenesis factor ► Primary and secondary piRNA biogenesis feed into a common biogenesis step ► Piwi and Aub, but not AGO3 are loaded with primary piRNAs

SummaryThe development of air-filled respiratory organs is crucial for survival at birth. We used a combination of live imaging and genetic analysis to dissect respiratory organ maturation in the embryonic Drosophila trachea. We found that tracheal tube maturation entails three precise epithelial transitions. Initially, a secretion burst deposits proteins into the lumen. Solid luminal material is then rapidly cleared from the tubes, and shortly thereafter liquid is removed. To elucidate the cellular mechanisms behind these transitions, we identified gas-filling-deficient mutants showing narrow or protein-clogged tubes. These mutations either disrupt endoplasmatic reticulum-to-Golgi vesicle transport or endocytosis. First, Sar1 is required for protein secretion, luminal matrix assembly, and diametric tube expansion. Subsequently, a sharp pulse of Rab5-dependent endocytic activity rapidly internalizes and clears luminal contents. The coordination of luminal matrix secretion and endocytosis may be a general mechanism in tubular organ morphogenesis and maturation.

The repression of transposable elements in eukaryotes often involves their transcriptional silencing via targeted chromatin modifications. In animal gonads, nuclear Argonaute proteins of the PIWI clade complexed with small guide RNAs (piRNAs) serve as sequence specificity determinants in this process. How binding of nuclear PIWI–piRNA complexes to nascent transcripts orchestrates heterochromatin formation and transcriptional silencing is unknown. Here, we characterize CG9754/Silencio as an essential piRNA pathway factor that is required for Piwi-mediated transcriptional silencing in Drosophila. Ectopic targeting of Silencio to RNA or DNA is sufficient to elicit silencing independently of Piwi and known piRNA pathway factors. Instead, Silencio requires the H3K9 methyltransferase Eggless/SetDB1 for its silencing ability. In agreement with this, SetDB1, but not Su(var)3-9, is required for Piwi-mediated transcriptional silencing genome-wide. Due to its interaction with the target-engaged Piwi–piRNA complex, we suggest that Silencio acts as linker between the sequence specificity factor Piwi and the cellular heterochromatin machinery.

PIWI clade Argonaute proteins silence transposon expression in animal gonads. Their target specificity is defined by bound ∼23- to 30-nucleotide (nt) PIWI-interacting RNAs (piRNAs) that are processed from single-stranded precursor transcripts via two distinct pathways. Primary piRNAs are defined by the endonuclease Zucchini, while biogenesis of secondary piRNAs depends on piRNA-guided transcript cleavage and results in piRNA amplification. Here, we analyze the interdependencies between these piRNA biogenesis pathways in developing Drosophila ovaries. We show that secondary piRNA-guided target slicing is the predominant mechanism that specifies transcripts—including those from piRNA clusters—as primary piRNA precursors and defines the spectrum of Piwi-bound piRNAs in germline cells. Post-transcriptional silencing in the cytoplasm therefore enforces nuclear transcriptional target silencing, which ensures the tight suppression of transposons during oogenesis. As target slicing also defines the nuclear piRNA pool during mouse spermatogenesis, our findings uncover an unexpected conceptual similarity between the mouse and fly piRNA pathways.

SummaryThe function of tubular epithelial organs like the kidney and lung is critically dependent on the length and diameter of their constituting branches. Genetic analysis of tube size control during Drosophila tracheal development has revealed that epithelial septate junction (SJ) components and the dynamic chitinous luminal matrix coordinate tube growth. However, the underlying molecular mechanisms controlling tube expansion so far remained elusive. Here, we present the analysis of two luminal chitin binding proteins with predicted polysaccharide deacetylase activities (ChLDs). ChLDs are required to assemble the cable-like extracellular matrix (ECM) and restrict tracheal tube elongation. Overexpression of native, but not of mutated, ChLD versions also interferes with the structural integrity of the intraluminal ECM and causes aberrant tube elongation. Whereas ChLD mutants have normal SJ structure and function, the luminal deposition of the ChLD requires intact cellular SJs. This identifies a new molecular function for SJs in the apical secretion of ChLD and positions ChLD downstream of the SJs in tube length control. The deposition of the chitin luminal matrix first promotes and coordinates radial tube expansion. We propose that the subsequent structural modification of chitin by chitin binding deacetylases selectively instructs the termination of tube elongation to the underlying epithelium.

Photoreceptors (R cells) in the Drosophila retina connect to targets in three distinct layers of the optic lobe of the brain: R1–R6 connect to the lamina, and R7 and R8 connect to distinct layers in the medulla [1]. In each of these layers, R axon termini are arranged in evenly spaced topographic arrays. In a genetic screen for mutants with abnormal R cell connectivity, we recovered mutations in flamingo (fmi). fmi encodes a seven-transmembrane cadherin, previously shown to function in planar cell polarity [2] and in dendritic patterning [3]. Here, we show that fmi has two specific functions in R8 axon targeting: it facilitates competitive interactions between adjacent R8 axons to ensure their correct spacing, and it promotes the formation of stable connections between R8 axons and their target cells in the medulla. The former suggests a general role for Fmi in establishing nonoverlapping dendritic and axonal target fields. The latter, together with the finding that N-Cadherin has an analogous role in R7 axon-target interactions [4], points to a cadherin-based system for target layer specificity in the Drosophila visual system [1].

Map-based positional cloning of Drosophila melanogaster genes is hampered by both the time-consuming, error-prone nature of traditional methods for genetic mapping and the difficulties in aligning the genetic and cytological maps with the genome sequence. The identification of sequence polymorphisms in the Drosophila genome will make it possible to map mutations directly to the genome sequence with high accuracy and resolution. Here we report the identification of 7,223 single-nucleotide polymorphisms (SNPs) and 1,392 insertions/deletions (InDels) in common laboratory strains of Drosophila. These sequence polymorphisms define a map of 787 autosomal marker loci with a resolution of 114 kb. We have established PCR product-length polymorphism (PLP) or restriction fragment-length polymorphism (RFLP) assays for 215 of these markers. We demonstrate the use of this map by delimiting two mutations to intervals of 169 kb and 307 kb, respectively. Using a local high-density SNP map, we also mapped a third mutation to a resolution of approximately 2 kb, sufficient to localize the mutation within a single gene. These methods should accelerate the rate of positional cloning in Drosophila.

Photoreceptors in the Drosophila eye project their axons retinotopically to targets in the optic lobe of the brain. The axons of photoreceptor cells R1-R6 terminate in the first optic ganglion, the lamina, while R7 and R8 axons project through the lamina to terminate in distinct layers of the second ganglion, the medulla. Here we report the identification of the gene brakeless (bks) and show that its function is required in the developing eye specifically for the lamina targeting of R1-R6 axons. In mosaic animals lacking bks function in the eye, R1-R6 axons project through the lamina to terminate in the medulla. Other aspects of visual system development appear completely normal: photoreceptor and lamina cell fates are correctly specified, R7 axons correctly target the medulla, and both correctly targeted R7 axons and mistargeted R1-R6 axons maintain their retinotopic order with respect to both anteroposterior and dorsoventral axes. bks encodes two unusually hydrophilic nuclear protein isoforms, one of which contains a putative C2H2 zinc finger domain. Transgenic expression of either Bks isoform is sufficient to restore the lamina targeting of R1-R6 axons in bks mosaics, but not to retarget R7 or R8 axons to the lamina. These data demonstrate the existence of a lamina-specific targeting mechanism for R1-R6 axons in the Drosophila visual system, and provide the first entry point in the molecular characterization of this process.

Members of the Hedgehog (Hh) family of secreted signaling proteins function as potent short-range organizers in animal development. Their range of action is limited by a C-terminal cholesterol tether and the upregulation of Patched (Ptc) receptor levels. Here we identify a novel segment-polarity gene in Drosophila, dispatched (disp), and demonstrate that its product is required in sending cells for normal Hh function. In the absence of Disp, cholesterol-modified but not cholesterol-free Hh is retained in these cells, indicating that Disp functions to release cholesterol-anchored Hh. Despite their opposite roles, Disp and Ptc share structural homology in the form of a sterol-sensing domain, suggesting that release and sequestration of cholesterol-modified Hh may be based on related molecular pathways.

Forward genetic screens in model organisms are an attractive means to identify those genes involved in any complex biological process, including neural circuit assembly. Although mutagenesis screens are readily performed to saturation, gene identification rarely is, being limited by the considerable effort generally required for positional cloning. Here, we apply a systematic positional cloning strategy to identify many of the genes required for neuronal wiring in the Drosophila visual system. From a large-scale forward genetic screen selecting for visual system wiring defects with a normal retinal pattern, we recovered 122 mutations in 42 genetic loci. For 6 of these loci, the underlying genetic lesions were previously identified using traditional methods. Using SNP-based mapping approaches, we have now identified 30 additional genes. Neuronal phenotypes have not previously been reported for 20 of these genes, and no mutant phenotype has been previously described for 5 genes. The genes encode a variety of proteins implicated in cellular processes such as gene regulation, cytoskeletal dynamics, axonal transport, and cell signalling. We conducted a comprehensive phenotypic analysis of 35 genes, scoring wiring defects according to 33 criteria. This work demonstrates the feasibility of combining large-scale gene identification with large-scale mutagenesis in Drosophila, and provides a comprehensive overview of the molecular mechanisms that regulate visual system wiring., Author Summary In the nervous system, every neuronal process should know where to grow and when to establish contacts to the next-order neurons. During development, it is known that neural circuit formation is primarily determined by the genes. To identify these genes, we focused on the Drosophila visual circuitry as a model system, and disrupted the genes randomly. From over 30,000 of these mutants, we found more than 100 mutants which have disrupted patterns of neural circuitry, which we assessed as representing about 40 genes. We have successfully nailed down which gene is disrupted in 36 of them. We provide a list of all of the genes we identified. Altogether, we performed a detailed characterization of the 35 mutant phenotypes, to assess which aspects of neural circuit formation are disrupted in each of the mutants. Summarizing and categorizing the phenotypic fingerprints of each mutant, we could see which genes are more closely related to the others. These data will be useful for clarifying the genetic program that controls neural circuit formation, not only for the Drosophila visual system, but also generally for nervous systems across the species.

BACKGROUND: Tube expansion defects like stenoses and atresias cause devastating human diseases. Luminal expansion during organogenesis begins to be elucidated in several systems but we still lack a mechanistic view of the process in many organs. The Drosophila tracheal respiratory system provides an amenable model to study tube size regulation. In the trachea, COPII anterograde transport of luminal proteins is required for extracellular matrix assembly and the concurrent tube expansion. PRINCIPAL FINDINGS: We identified and analyzed Drosophila COPI retrograde transport mutants with narrow tracheal tubes. gammaCOP mutants fail to efficiently secrete luminal components and assemble the luminal chitinous matrix during tracheal tube expansion. Likewise, tube extension is defective in salivary glands, where it also coincides with a failure in the luminal deposition and assembly of a distinct, transient intraluminal matrix. Drosophila gammaCOP colocalizes with cis-Golgi markers and in gammaCOP mutant embryos the ER and Golgi structures are severely disrupted. Analysis of gammaCOP and Sar1 double mutants suggests that bidirectional ER-Golgi traffic maintains the ER and Golgi compartments and is required for secretion and assembly of luminal matrixes during tube expansion. CONCLUSIONS/SIGNIFICANCE: Our results demonstrate the function of COPI components in organ morphogenesis and highlight the common role of apical secretion and assembly of transient organotypic matrices in tube expansion. Intraluminal matrices have been detected in the notochord of ascidians and zebrafish COPI mutants show defects in notochord expansion. Thus, the programmed deposition and growth of distinct luminal molds may provide distending forces during tube expansion in diverse organs.