RNA interference (RNAi) in tissue culture cells has emerged as an excellent methodology for identifying gene functions systematically and in an unbiased manner. Here, we describe how RNAi high-throughput screening (HTS) in Drosophila cells are currently being performed and emphasize the strengths and weaknesses of the approach. Further, to demonstrate the versatility of the technology, we provide examples of the various applications of the method to problems in signal transduction and cell and developmental biology. Finally, we discuss emerging technological advances that will extend RNAi-based screening methods.
With the development of genome-wide RNAi libraries, it is now possible to screen for novel components of mitogen-activated protein kinase (MAPK) pathways in cell culture. Although genetic dissection in model organisms and biochemical approaches in mammalian cells have been successful in identifying the core signaling cassettes of these pathways, high-throughput assays can yield unbiased, functional genomic insight into pathway regulation. We describe general high-throughput approaches to assaying MAPK signaling and the receptor tyrosine kinase (RTK)/extracellular signal-regulated kinase (ERK) pathway in particular using a phospho-specific antibody-based readout of pathway activity. We also provide examples of secondary validation screens and methods for managing large datasets for future in vivo functional characterization.
RNA interference has re-energized the field of functional genomics by enabling genome-scale loss-of-function screens in cultured cells. Looking back on the lessons that have been learned from the first wave of technology developments and applications in this exciting field, we provide both a user's guide for newcomers to the field and a detailed examination of some more complex issues, particularly concerning optimization and quality control, for more advanced users. From a discussion of cell lines, screening paradigms, reagent types and read-out methodologies, we explore in particular the complexities of designing optimal controls and normalization strategies for these challenging but extremely powerful studies.
Large-scale RNA interference (RNAi)-based analyses, very much as other 'omic' approaches, have inherent rates of false positives and negatives. The variability in the standards of care applied to validate results from these studies, if left unchecked, could eventually begin to undermine the credibility of RNAi as a powerful functional approach. This Commentary is an invitation to an open discussion started among various users of RNAi to set forth accepted standards that would insure the quality and accuracy of information in the large datasets coming out of genome-scale screens.
Wnts [also known as Wingless (Wg)] are a family of conserved signaling molecules involved in a plethora of fundamental developmental and cell biological processes, such as cell proliferation, differentiation, and cell polarity. Dysregulation of the pathway can be detrimental, because several components are tumorigenic when mutated and are associated with hepatic, colorectal, breast, and skin cancers. First identified in the fruit fly Drosophila melanogaster as a gene family responsible for patterning the embryonic epidermis, the Wnt gene family, including Wg, encode secreted glycoproteins that activate receptor-mediated signaling pathways leading to numerous transcriptional and cellular responses. The main function of the canonical Wg pathway is to stabilize the cytoplasmic pool of a key mediator, beta-catenin [beta-catenin, known as Armadillo (Arm) in fruit flies], which is otherwise degraded by the proteasome pathway. Initially identified as a key player in stabilizing cell-cell adherens junctions, Arm is now known to also act as a transcription factor by forming a complex with the lymphoid enhancer factor (LEF)/T cell-specific transcription factor (TCF) family of high mobility group (HMG)-box transcription factors. Upon Wnt/Wg stimulation, stabilized Arm translocates to the nucleus, where, together with LEF/TCF transcription factors, it activates downstream target genes that regulate numerous cell biological processes.
Pattern formation during development is controlled to a great extent by a small number of conserved signal transduction pathways that are activated by extracellular ligands such as Hedgehog, Wingless or Decapentaplegic. Genetic experiments have identified heparan sulphate proteoglycans (HSPGs) as important regulators of the tissue distribution of these extracellular signalling molecules. Several recent reports provide important new insights into the mechanisms by which HSPGs function during development.
This chapter describes the method used to conduct high-throughput screening (HTs) by RNA interference in Drosophila tissue culture cells. It covers four main topics: (1) a brief description of the existing platforms to conduct RNAi-screens in cell-based assays; (2) a table of the Drosophila cell lines available for these screens and a brief mention of the need to establish other cell lines as well as cultures of primary cells; (3) a discussion of the considerations and protocols involved in establishing assays suitable for HTS in a 384-well format; and (A) a summary of the various ways of handling raw data from an ongoing screen, with special emphasis on how to apply normalization for experimental variation and statistical filters to sort out noise from signals.
The increasing prevalence of obesity and other nutrition-related chronic diseases has prompted considerable efforts to understand their pathogenesis and treatment. One experimental approach is to overexpress, inactivate, or manipulate specific genes that regulate energy metabolism and fat storage. Many such techniques are fully established, routine tools in Drosophila and C. elegans, which provide elegant models for dissecting endocrine problems and metabolic pathways.
The availability of complete genome sequences from many organisms has yielded the ability to perform high-throughput, genome-wide screens of gene function. Within the past year, rapid advances have been made towards this goal in many major model systems, including yeast, worms, flies, and mammals. Yeast genome-wide screens have taken advantage of libraries of deletion strains, but RNA-interference has been used in other organisms to knockdown gene function. Examples of recent large-scale functional genetic screens include drug-target identification in yeast, regulators of fat accumulation in worms, growth and viability in flies, and proteasome-mediated degradation in mammalian cells. Within the next five years, such screens are likely to lead to annotation of function of most genes across multiple organisms. Integration of such data with other genomic approaches will extend our understanding of cellular networks.
Innate immune responses are mediated by the activation of various signaling processes. Here, we describe our current knowledge on Janus kinase (JAK)/signal transducers and activators of transcription (STAT) signaling in the Drosophila immune response. First, we briefly introduce the main effectors involved in the humoral and cellular responses, such as anti-bacterial peptides and hemocytes. Second, we describe the canonical JAK/STAT-signaling pathway, as established from extensive studies in mammalian systems, and we introduce the Drosophila components of the JAK/STAT pathway, as discovered from studies on embryonic development. Third, we describe the various roles of JAK/STAT signaling in both humoral and cellular responses. We present the JAK/STAT-dependent humoral factors, such as the thioester-containing proteins and the Tot peptides, produced by the fat body in response to septic injury. We also discuss the possible involvement of the JAK/STAT pathway in cellular responses, including hemocyte proliferation and differentiation. Finally, we present how cytokines, such as Upd3, might contribute to the integration of the immune responses at the organism level by orchestrating the response of various immune cells and organs, such as fat body, hemocytes, and lymph glands.
The completion of whole-genome sequencing of various model organisms and the recent explosion of new technologies in the field of Functional Genomics and Proteomics is poised to revolutionize the way scientists identify and characterize gene function. One of the most significant advances in recent years has been the application of RNA interference (RNAi) as a means of assaying gene function. In the post-genomic era, advances in the field of cancer biology will rely upon the rapid identification and characterization of genes that regulate cell growth, proliferation, and apoptosis. Significant efforts are being directed towards cancer therapy and devising efficient means of selectively delivering drugs to cancerous cells. In this review, we discuss the promise of integrating genome-wide RNAi screens with proteomic approaches and small-molecule chemical genetic screens, towards improving our ability to understand and treat cancer.
The structure and function of epithelial sheets generally depend on apicobasal polarization, which is achieved and maintained by linking asymmetrically distributed intercellular junctions to the cytoskeleton of individual cells. Recent studies in both Drosophila and vertebrate epithelia have yielded new insights into the conserved mechanisms by which apicobasal polarity is established and maintained during development. In mature polarized epithelia, apicobasal polarity is important for the establishment of adhesive junctions and the formation of a paracellular diffusion barrier that prevents the movement of solutes across the epithelium. Recent findings show that segregation of ligand and receptor with one on each side of this barrier can be a crucial regulator of cell-cell signaling events.
In the last century, genetics has developed into one of the most powerful tools for addressing basic questions concerning inheritance, development, individual and social operations and death. Here we summarize the current approaches to these questions in four of the most advanced models organisms: Saccharomyces cerevisiae (yeast), Caenorhabditis elegans (worm), Drosophila melanogaster (fly) and Mus musculus (mouse). The genomes of each of these four models have been sequenced, and all have well developed methods of efficient genetic manipulations.
The formation of complex patterns in multi-cellular organisms is regulated by a number of signaling pathways. In particular, the Wnt and Hedgehog (Hh) pathways have been identified as critical organizers of pattern in many tissues. Although extensive biochemical and genetic studies have elucidated the central components of the signal transduction pathways regulated by these secreted molecules, we still do not understand fully how they organize gradients of gene activities through field of cells. Studies in Drosophila have implicated a role for heparan sulfate proteoglycans (HSPGs) in regulating the signaling activities and distribution of both Wnt and Hh. Here we review these findings and discuss various models by which HSPGs regulate the distributions of Wnt and Hh morphogens.
The Hedgehog (Hh) family of signaling molecules are key agents in patterning numerous types of tissues. Mutations in Hh and its downstream signaling molecules are also associated with numerous oncogenic and disease states. Consequently, understanding the mechanisms by which Hh signals are transduced is important for understanding both development and disease. Recent studies have clarified several aspects of Hh signal transduction. Several new Sonic Hedgehog binding partners have been identified. Cholesterol and palmitic acid modifications of Hh and Sonic hedgehog have been examined in greater detail. Characterization of the trafficking patterns of the Patched and Smoothened proteins has demonstrated that these two proteins function very differently from the previously established models. The Fused kinase has been demonstrated to phosphorylate the kinesin-like protein Costal2 and the sites identified, while Cubitus interruptus has been shown to be phosphorylated in a hierarchical manner by three different kinases. Finally, the interactions, both genetic and physical, between Fused, Costal2, Cubitus interruptus, and Suppressor of Fused have been further elucidated.
Heparan sulphate proteoglycans (HSPG's) are cell surface proteins to which long, unbranched chains of modified sugars called heparan sulphate glycosaminoglycans have been covalently attached. Cell culture studies have demonstrated that HSPG's are required for optimal signal transduction by many secreted cell signaling molecules. Now, genetic studies in both Drosophila and vertebrates have illustrated that HSPG's play important roles in signal transduction in vivo and have also begun to reveal new roles for HSPG's in signaling events. In particular, HSPG's have been shown to be important in ligand sequestration of wingless, for the transport of the Hedgehog ligand, and for modulation of the Dpp morphogenetic gradient.
The JAK/STAT pathway was originally identified in mammals. Studies of this pathway in the mouse have revealed that JAK/STAT signaling plays a central role during hematopoeisis and other developmental processes. The role of JAK/STAT signaling in blood appears to be conserved throughout evolution, as it is also required during fly hematopoeisis. Studies in Dictyostelium, Drosophila, and zebrafish have shown that the JAK/STAT pathway is also required in an unusually broad set of developmental decisions, including cell proliferation, cell fate determination, cell migration, planar polarity, convergent extension, and immunity. There is increasing evidence that the versatility of this pathway relies on its cooperation with other signal transduction pathways. In this review, we discuss the components of the JAK/STAT pathway in model organisms and what is known about its requirement in cellular and developmental processes. In particular, we emphasize recent insights into the role that this pathway plays in the control of cell movement.
Epithelial morphogenesis comprises the various processes by which epithelia contribute to organ formation and body shape. These complex and diverse events play a central role in animal development and regeneration. Recently, the characterization of some of the molecular mechanisms involved in epithelial morphogenesis has provided an abundance of new information on the role and regulation of the cytoskeleton, cell-cell adhesion, and cell-matrix adhesion in these processes. In this review, we discuss our current understanding of the molecular mechanisms driving cell shape changes, cell intercalation, fusion of epithelia, ingression, egression, and cell migration. Our discussion is mostly focused on results from Drosophila and mammalian tissue culture but also draws on the insights gained from other organisms.