Drosophila genome-wide RNAi screens: are they delivering the promise?

The emergence of RNA interference (RNAi) on the heels of the successful completion of the Drosophila genome project was seen by many as the ace in functional genomics: Its application would quickly assign a function to all genes in this organism and help delineate the complex web of interactions or networks linking them at the systemic level. A few years wiser and a number of genome-wide Drosophila RNAi screens later, we reflect on the state of high-throughput RNAi screens in Drosophila and ask whether the initial promise was fulfilled. We review the impact that this approach has had in the field of Drosophila research and chart out strategies to extract maximal benefit from the application of RNAi to gene discovery and pursuit of systems biology.

Towards automated cellular image segmentation for RNAi genome-wide screening.

The Rho family of small GTPases is essential for morphological changes during normal cell development and migration, as well as during disease states such as cancer. Our goal is to identify novel effectors of Rho proteins using a cell-based assay for Rho activity to perform genome-wide functional screens using double stranded RNA (dsRNAs) interference. We aim to discover genes could cause the cell phenotype changed dramatically. Biologists currently attempt to perform the genome-wide RNAi screening to identify various image phenotypes. RNAi genome-wide screening, however, could easily generate more than a million of images per study, manual analysis is thus prohibitive. Image analysis becomes a bottleneck in realizing high content imaging screens. We propose a two-step segmentation approach to solve this problem. First, we determine the center of a cell using the information in the DNA-channel by segmenting the DNA nuclei and the dissimilarity function is employed to attenuate the over-segmentation problem, then we estimate a rough boundary for each cell using a polygon. Second, we apply fuzzy c-means based multi-threshold segmentation and sharpening technology; for isolation of touching spots, marker-controlled watershed is employed to remove touching cells. Furthermore, Voronoi diagrams are employed to correct the segmentation errors caused by overlapping cells. Image features are extracted for each cell. K-nearest neighbor classifier (KNN) is employed to perform cell phenotype classification. Experimental results indicate that the proposed approach can be used to identify cell phenotypes of RNAi genome-wide screens.

A functional genomic analysis of cell morphology using RNA interference.

BACKGROUND: The diversity of metazoan cell shapes is influenced by the dynamic cytoskeletal network. With the advent of RNA-interference (RNAi) technology, it is now possible to screen systematically for genes controlling specific cell-biological processes, including those required to generate distinct morphologies. RESULTS: We adapted existing RNAi technology in Drosophila cell culture for use in high-throughput screens to enable a comprehensive genetic dissection of cell morphogenesis. To identify genes responsible for the characteristic shape of two morphologically distinct cell lines, we performed RNAi screens in each line with a set of double-stranded RNAs (dsRNAs) targeting 994 predicted cell shape regulators. Using automated fluorescence microscopy to visualize actin filaments, microtubules and DNA, we detected morphological phenotypes for 160 genes, one-third of which have not been previously characterized in vivo. Genes with similar phenotypes corresponded to known components of pathways controlling cytoskeletal organization and cell shape, leading us to propose similar functions for previously uncharacterized genes. Furthermore, we were able to uncover genes acting within a specific pathway using a co-RNAi screen to identify dsRNA suppressors of a cell shape change induced by Pten dsRNA. CONCLUSIONS: Using RNAi, we identified genes that influence cytoskeletal organization and morphology in two distinct cell types. Some genes exhibited similar RNAi phenotypes in both cell types, while others appeared to have cell-type-specific functions, in part reflecting the different mechanisms used to generate a round or a flat cell morphology.

Drosophila Stardust interacts with Crumbs to control polarity of epithelia but not neuroblasts.

Establishing cellular polarity is critical for tissue organization and function. Initially discovered in the landmark genetic screen for Drosophila developmental mutants, bazooka, crumbs, shotgun and stardust mutants exhibit severe disruption in apicobasal polarity in embryonic epithelia, resulting in multilayered epithelia, tissue disintegration, and defects in cuticle formation. Here we report that stardust encodes single PDZ domain MAGUK (membrane-associated guanylate kinase) proteins that are expressed in all primary embryonic epithelia from the onset of gastrulation. Stardust colocalizes with Crumbs at the apicolateral boundary, although their expression patterns in sensory organs differ. Stardust binds to the carboxy terminus of Crumbs in vitro, and Stardust and Crumbs are mutually dependent in their stability, localization and function in controlling the apicobasal polarity of epithelial cells. However, for the subset of ectodermal cells that delaminate and form neuroblasts, their polarity requires the function of Bazooka, but not of Stardust or Crumbs.

Spatial control of the actin cytoskeleton in Drosophila epithelial cells.

The actin cytoskeleton orders cellular space and transduces many of the forces required for morphogenesis. Here we combine genetics and cell biology to identify genes that control the polarized distribution of actin filaments within the Drosophila follicular epithelium. We find that profilin and cofilin regulate actin-filament formation throughout the cell cortex. In contrast, CAP-a Drosophila homologue of Adenylyl Cyclase Associated Proteins-functions specifically to limit actin-filament formation catalysed by Ena at apical cell junctions. The Abl tyrosine kinase also collaborates in this process. We therefore propose that CAP, Ena and Abl act in concert to modulate the subcellular distribution of actin filaments in Drosophila.

Investigation of leading edge formation at the interface of amnioserosa and dorsal ectoderm in the Drosophila embryo.

The leading edge (LE) is a single row of cells in the Drosophila embryonic epidermis that marks the boundary between two fields of cells: the amnioserosa and the dorsal ectoderm. LE cells play a crucial role in the morphogenetic process of dorsal closure and eventually form the dorsal midline of the embryo. Mutations that block LE differentiation result in a failure of dorsal closure and embryonic lethality. How LE cells are specified remains unclear. To explore whether LE cells are specified in response to early dorsoventral patterning information or whether they arise secondarily, we have altered the extent of amnioserosa and dorsal ectoderm genetically, and assayed LE cell fate. We did not observe an expansion of LE fate in dorsalized or ventralized mutants. Furthermore, we observed that the LE fate arises as a single row of cells, wherever amnioserosa tissue and dorsal epidermis are physically juxtaposed. Taken together our data indicate that LE formation is a secondary consequence of early zygotic dorsal patterning signals. In particular, proper LE specification requires the function of genes such as u-shaped and hindsight, which are direct transcriptional targets of the early Decapentaplegic/Screw patterning gradient, to establish a competency zone from which LE arises. We propose that subsequent inductive signaling between amnioserosa and dorsal ectoderm restricts the formation of LE to a single row of cells.

The Drosophila JNK pathway controls the morphogenesis of the egg dorsal appendages and micropyle.

During Drosophila oogenesis, the formation of the egg respiratory appendages and the micropyle require the shaping of anterior and dorsal follicle cells. Prior to their morphogenesis, cells of the presumptive appendages are determined by integrating dorsal-ventral and anterior-posterior positional information provided by the epidermal growth factor receptor (EGFR) and Decapentaplegic (Dpp) pathways, respectively. We show here that another signaling pathway, the Drosophila Jun-N-terminal kinase (JNK) cascade, is essential for the correct morphogenesis of the dorsal appendages and the micropyle during oogenesis. Mutant follicle cell clones of members of the JNK pathway, including DJNKK/hemipterous (hep), DJNK/basket (bsk), and Djun, block dorsal appendage formation and affect the micropyle shape and size, suggesting a late requirement for the JNK pathway in anterior chorion morphogenesis. In support of this view, hep does not affect early follicle cell patterning as indicated by the normal expression of kekkon (kek) and Broad-Complex (BR-C), two of the targets of the EGFR pathway in dorsal follicle cells. Furthermore, the expression of the TGF-beta homolog dpp, which is under the control of hep in embryos, is not coupled to JNK activity during oogenesis. We show that hep controls the expression of puckered (puc) in the follicular epithelium in a cell-autonomous manner. Since puc overexpression in the egg follicular epithelium mimics JNK appendages and micropyle phenotypes, it indicates a negative role of puc in their morphogenesis. The role of the JNK pathway in the morphogenesis of follicle cells and other epithelia during development is discussed.

Dual role of the fringe connection gene in both heparan sulphate and fringe-dependent signalling events.

The precise regulation of growth factor signalling is crucial to the molecular control of development in Drosophila. Post-translational modification of signalling molecules is one of the mechanisms that modulate developmental signalling specificity. We describe a new gene, fringe connection (frc), that encodes a nucleotide-sugar transporter that transfers UDP-glucuronic acid, UDP-N-acetylglucosamine and possibly UDP-xylose from the cytoplasm into the lumen of the endoplasmic reticulum/Golgi. Embryos with the frc mutation display defects in Wingless, Hedgehog and fibroblast growth factor signalling. Clonal analysis shows that fringe-dependent Notch signalling is disrupted in frc mutant tissue.

The cell adhesion molecule Echinoid defines a new pathway that antagonizes the Drosophila EGF receptor signaling pathway.

Photoreceptor and cone cells in the Drosophila eye are recruited following activation of the epidermal growth factor receptor (EGFR) pathway. We have identified echinoid (ed) as a novel putative cell adhesion molecule that negatively regulates EGFR signaling. The ed mutant phenotype is associated with extra photoreceptor and cone cells. Conversely, ectopic expression of ed in the eye leads to a reduction in the number of photoreceptor cells. ed expression is independent of EGFR signaling and ED is localized to the plasma membrane of every cells throughout the eye disc. We present evidence that ed acts nonautonomously to generate extra R7 cells by a mechanism that is sina-independent but upstream of Tramtrack (TTK88). Together, our results support a model whereby ED defines an independent pathway that antagonizes EGFR signaling by regulating the activity, but not the level, of the TTK88 transcriptional repressor.

Heparan sulfate proteoglycans are critical for the organization of the extracellular distribution of Wingless.

Recent studies in Drosophila have shown that heparan sulfate proteoglycans (HSPGs) are required for Wingless (Wg/Wnt) signaling. In addition, genetic and phenotypic analyses have implicated the glypican gene dally in this process. Here, we report the identification of another Drosophila glypican gene, dally-like (dly) and show that it is also involved in Wg signaling. Inhibition of dly gene activity implicates a function for DLY in Wg reception and we show that overexpression of DLY leads to an accumulation of extracellular Wg. We propose that DLY plays a role in the extracellular distribution of Wg. Consistent with this model, a dramatic decrease of extracellular Wg was detected in clones of cells that are deficient in proper glycosaminoglycan biosynthesis. We conclude that HSPGs play an important role in organizing the extracellular distribution of Wg.

Multiple roles for four-jointed in planar polarity and limb patterning.

Insect cuticles have been a model system for the study of planar polarity for many years and a number of genes required for this process have been identified. These genes organise the polarised arrangement of hairs on the legs, wings, thorax, and abdomen of adult Drosophila. It has previously been shown that four-jointed is involved in planar polarity decisions in the eye as well as proximal distal leg and wing development. We now present evidence that four-jointed is expressed in a gradient through the developing wing and show that it is required for planar polarity determination in both the wing and the abdomen. Clones of cells either lacking or ectopically expressing four-jointed cause both autonomous and nonautonomous repolarisation of hairs in these tissues. We propose that the inferred four-jointed expression gradient is important for planar polarity establishment and that local inversions of the gradient by the clones are the probable cause of the observed polarity phenotypes. In addition we observe defects in wing vein development. The subtle phenotypes of mutant flies, and the diverse patterning processes in which it is involved, suggest that four-jointed may act as a modifier of the activity of multiple other signalling factors.

Presenilin affects arm/beta-catenin localization and function in Drosophila.

Presenilin is an essential gene for development that when disrupted leads to a neurogenic phenotype that closely resembles Notch loss of function in Drosophila. In humans, many naturally occurring mutations in Presenilin 1 or 2 cause early onset Alzheimer's disease. Both loss of expression and overexpression of Presenilin suggested a role for this protein in the localization of Armadillo/beta-catenin. In blastoderm stage Presenilin mutants, Arm is aberrantly distributed, often in Ubiquitin-immunoreactive cytoplasmic inclusions predominantly located basally in the cell. These inclusions were not observed in loss of function Notch mutants, suggesting that failure to process Notch is not the only consequence of the loss of Presenilin function. Human presenilin 1 expressed in Drosophila produces embryonic phenotypes resembling those associated with mutations in Armadillo and exhibited reduced Armadillo at the plasma membrane that is likely due to retention of Armadillo in a complex with Presenilin. The interaction between Armadillo/beta-catenin and Presenilin 1 requires a third protein which may be delta-catenin. Our results suggest that Presenilin may regulate the delivery of a multiprotein complex that regulates Armadillo trafficking between the adherens junction and the proteasome.

Identification of autosomal regions involved in Drosophila Raf function.

Raf is an essential downstream effector of activated p21(Ras) (Ras) in transducing proliferation or differentiation signals. Following binding to Ras, Raf is translocated to the plasma membrane, where it is activated by a yet unidentified "Raf activator." In an attempt to identify the Raf activator or additional molecules involved in the Raf signaling pathway, we conducted a genetic screen to identify genomic regions that are required for the biological function of Drosophila Raf (Draf). We tested a collection of chromosomal deficiencies representing approximately 70% of the autosomal euchromatic genomic regions for their abilities to enhance the lethality associated with a hypomorphic viable allele of Draf, Draf(Su2). Of the 148 autosomal deficiencies tested, 23 behaved as dominant enhancers of Draf(Su2), causing lethality in Draf(Su2) hemizygous males. Four of these deficiencies identified genes known to be involved in the Drosophila Ras/Raf (Ras1/Draf) pathway: Ras1, rolled (rl, encoding a MAPK), 14-3-3epsilon, and bowel (bowl). Two additional deficiencies removed the Drosophila Tec and Src homologs, Tec29A and Src64B. We demonstrate that Src64B interacts genetically with Draf and that an activated form of Src64B, when overexpressed in early embryos, causes ectopic expression of the Torso (Tor) receptor tyrosine kinase-target gene tailless. In addition, we show that a mutation in Tec29A partially suppresses a gain-of-function mutation in tor. These results suggest that Tec29A and Src64B are involved in Tor signaling, raising the possibility that they function to activate Draf. Finally, we discovered a genetic interaction between Draf(Su2) and Df(3L)vin5 that revealed a novel role of Draf in limb development. We find that loss of Draf activity causes limb defects, including pattern duplications, consistent with a role for Draf in regulation of engrailed (en) expression in imaginal discs.

A cyclase-associated protein regulates actin and cell polarity during Drosophila oogenesis and in yeast.

BACKGROUND: A polarised cytoskeleton is required to pattern cellular space, and for many aspects of cell behaviour. While the mechanisms ordering the actin cytoskeleton have been extensively studied in yeast, little is known about the analogous processes in other organisms. We have used Drosophila oogenesis as a model genetic system in which to investigate control of cytoskeletal organisation and cell polarity in multicellular eukaryotes. RESULTS: In a screen to identify genes required for Drosophila oocyte polarity, we isolated a Drosophila homologue of the yeast cyclase-associated protein, CAP. Here we show that CAP preferentially accumulates in the oocyte, where it inhibits actin polymerisation. CAP also has a role in oocyte polarity, as cap mutants fail to establish the proper, asymmetric distribution of mRNA determinants within the oocyte. Similarly in yeast, loss of CAP causes analogous polarity defects, altering the distribution of actin filaments and mRNA determinants. CONCLUSIONS: This study identifies CAP as a new effector of actin dynamics in Drosophila. As CAP controls the spatial distribution of actin filaments and mRNA determinants in both yeast and Drosophila, we conclude that CAP has an evolutionarily conserved function in the genesis of eukaryotic cell polarity.

Functional binding of secreted molecules to heparan sulfate proteoglycans in Drosophila.

Heparan sulfate proteoglycans (HSPGs) are associated with the cell surface and covalently linked to a small number of long unbranched chains of repeating disaccharides. Numerous biochemical studies of these extracellular matrix molecules have implicated them in a variety of biological phenomena, in particular cell-cell interactions. Recent genetic studies in Drosophila have begun to clarify the function of HSPGs in vivo and recent findings have implicated HSPGs in Wnt, Hedgehog, fibroblast growth factor and transforming growth factor-beta signaling pathways during development.

Sex determination: co-opted signals determine gender.

The Drosophila JAK-STAT pathway and its ligand Unpaired are required for a wide range of developmental processes. Recent results have identified Unpaired as an activator of sex-lethal and revealed a new role for the JAK-STAT pathway in sex determination.