RNA interference (RNAi) leads to sequence-specific knockdown of gene function. The approach can be used in large-scale screens to interrogate function in various model organisms and an increasing number of other species. Genome-scale RNAi screens are routinely performed in cultured or primary cells or in vivo in organisms such as C. elegans. High-throughput RNAi screening is benefitting from the development of sophisticated new instrumentation and software tools for collecting and analyzing data, including high-content image data. The results of large-scale RNAi screens have already proved useful, leading to new understandings of gene function relevant to topics such as infection, cancer, obesity and aging. Nevertheless, important caveats apply and should be taken into consideration when developing or interpreting RNAi screens. Some level of false discovery is inherent to high-throughput approaches and specific to RNAi screens, false discovery due to off-target effects (OTEs) of RNAi reagents remains a problem. The need to improve our ability to use RNAi to elucidate gene function at large scale and in additional systems continues to be addressed through improved RNAi library design, development of innovative computational and analysis tools and other approaches.
RNAi; high-throughput screens; high-content imaging; cell-based assays
RNA interference (RNAi) is an effective tool for genome-scale, high-throughput analysis of gene function. In the past five years, a number of genome-scale RNAi high-throughput screens (HTSs) have been done in both Drosophila and mammalian cultured cells to study diverse biological processes, including signal transduction, cancer biology, and host cell responses to infection. Results from these screens have led to the identification of new components of these processes and, importantly, have also provided insights into the complexity of biological systems, forcing new and innovative approaches to understanding functional networks in cells. Here, we review the main findings that have emerged from RNAi HTS and discuss technical issues that remain to be improved, in particular the verification of RNAi results and validation of their biological relevance. Furthermore, we discuss the importance of multiplexed and integrated experimental data analysis pipelines to RNAi HTS.
bioinformatics; cell biology; Drosophila; high-throughput screening
Originally identified as a response to starvation in yeast, autophagy is now understood to fulfill a variety of roles in higher eukaryotes, from the maintenance of cellular homeostasis to the cellular response to stress, starvation, and infection. Although genetics and biochemical studies in yeast have identified many components involved in autophagy, the findings that some of the essential components of the yeast pathway are missing in higher organisms underscore the need to study autophagy in more complex systems. This review focuses on the use of the fruitfly, Drosophila melanogaster as a model system for analysis of autophagy. Drosophila is an organism well-suited for genetic analysis and represents an intermediate between yeast and mammals with respect to conservation of the autophagy machinery. Furthermore, the complex biology and physiology of Drosophila presents an opportunity to model human diseases in a tissue specific and analogous context.
Drosophila; Autophagy; Atg; Model system
Current studies of physiological communication between Drosophila organs are beginning to address the fundamental problem of how nutrients regulate organismal growth, stem cell behavior, immunity, and aging. Advances in the Drosophila genetic tool kit will allow the design of genetic screens to systematically identify factors involved in organ communication.
Systems biology aims to describe the complex interplays between cellular building blocks which, in their concurrence, give rise to the emergent properties observed in cellular behaviors and responses. This approach tries to determine the molecular players and the architectural principles of their interactions within the genetic networks that control certain biological processes. Large-scale loss-of-function screens, applicable in various different model systems, have begun to systematically interrogate entire genomes to identify the genes that contribute to a certain cellular response. In particular, RNA interference (RNAi)-based high-throughput screens have been instrumental in determining the composition of regulatory systems and paired with integrative data analyses have begun to delineate the genetic networks that control cell biological and developmental processes. Through the creation of tools for both, in vitro and in vivo genome-wide RNAi screens, Drosophila melanogaster has emerged as one of the key model organisms in systems biology research and over the last years has massively contributed to and hence shaped this discipline.
Signaling proteins often form dynamic protein-protein interaction (PPI) complexes to achieve multi-functionality. Methods to abrogate a subset of PPI interfaces without depleting the full-length protein will be valuable for structure-function relationship annotations. Here, we describe the use of Peptide Aptamer Interference (PAPTi) approach for structure-function network studies. We identified peptide aptamers against Dishevelled (Dsh) and β-catenin (β-cat) to target the Wnt signaling pathway and demonstrate that these FN3-based MONOBODYs (FNDYs) can be used to perturb protein activities both in vitro and in vivo. Further, to investigate the crosstalk between the Wnt and Notch pathways, we isolated FNDYs against the Notch Ankyrin (ANK) region and demonstrate that perturbing the ANK domain of Notch increases the inhibitory activity of Notch towards Wnt signaling. Altogether, these studies demonstrate the power of the PAPTi approach to dissect specific PPI interactions within signaling networks.
Cytoplasmic Polyadenylation Element Binding (CPEB) proteins are translational regulators that can either activate or repress translation depending on the target mRNA and the specific biological context. There are two CPEB subfamilies and most animals have one or more genes from each. Drosophila has a single CPEB gene, orb and orb2, from each subfamily. orb expression is only detected at high levels in the germline and has critical functions in oogenesis but not spermatogenesis. By contrast, orb2 is broadly expressed in the soma; and previous studies have revealed important functions in asymmetric cell division, viability, motor function, learning, and memory. Here we show that orb2 is also expressed in the adult male germline and that it has essential functions in programming the progression of spermatogenesis from meiosis through differentiation. Like the translational regulators boule (bol) and off-schedule (ofs), orb2 is required for meiosis and orb2 mutant spermatocytes undergo a prolonged arrest during the meiotic G2-M transition. However, orb2 differs from boule and off-schedule in that this arrest occurs at a later step in meiotic progression after the synthesis of the meiotic regulator twine. orb2 is also required for the orderly differentiation of the spermatids after meiosis is complete. The differentiation defects in orb2 mutants include abnormal elongation of the spermatid flagellar axonemes, a failure in individualization and improper post-meiotic gene expression. Amongst the orb2 differentiation targets are orb and two other mRNAs, which are transcribed post-meiotically and localized to the tip of the flagellar axonemes. Additionally, analysis of a partial loss of function orb2 mutant suggests that the orb2 differentiation phenotypes are independent of the earlier arrest in meiosis.
Cytoplasmic Polyadenylation Element Binding (CPEB) proteins bind and recognize CPE sequences in the 3′ UTRs of target mRNAs and can activate and/or repress their translation depending on the mRNA species and the biological context. Drosophila has two CPEB family genes, orb and orb2. orb is expressed in the germline of both sexes and has critical functions at multiple steps during oogenesis; however, it plays only a limited role in spermatogenesis. Here we show that the second CPEB family gene orb2 has the opposite sex specificity in germline development. While it appears to be dispensable for oogenesis, orb2 has essential functions during spermatogenesis. It is required for programming the orderly and sequential progression of spermatogenesis from meiosis through differentiation. orb2 mutants fail to execute the meiotic G2-M transition and exhibit a range of defects in the process of sperm differentiation.
A Drosophila transgenic RNAi screen targeting the glycan genome, including all N/O/GAG-glycan biosynthesis/modification enzymes and glycan-binding lectins, was conducted to discover novel glycan functions in synaptogenesis. As proof-of-product, we characterized functionally paired heparan sulfate (HS) 6-O-sulfotransferase (hs6st) and sulfatase (sulf1), which bidirectionally control HS proteoglycan (HSPG) sulfation. RNAi knockdown of hs6st and sulf1 causes opposite effects on functional synapse development, with decreased (hs6st) and increased (sulf1) neurotransmission strength confirmed in null mutants. HSPG co-receptors for WNT and BMP intercellular signaling, Dally-like Protein and Syndecan, are differentially misregulated in the synaptomatrix of these mutants. Consistently, hs6st and sulf1 nulls differentially elevate both WNT (Wingless; Wg) and BMP (Glass Bottom Boat; Gbb) ligand abundance in the synaptomatrix. Anterograde Wg signaling via Wg receptor dFrizzled2 C-terminus nuclear import and retrograde Gbb signaling via synaptic MAD phosphorylation and nuclear import are differentially activated in hs6st and sulf1 mutants. Consequently, transcriptional control of presynaptic glutamate release machinery and postsynaptic glutamate receptors is bidirectionally altered in hs6st and sulf1 mutants, explaining the bidirectional change in synaptic functional strength. Genetic correction of the altered WNT/BMP signaling restores normal synaptic development in both mutant conditions, proving that altered trans-synaptic signaling causes functional differentiation defects.
Glycans are sugar additions to proteins. Surrounding all eukaryotic cells, secreted and membrane glycans form a glycocalyx that regulates cell–cell signaling. However, the mechanisms controlling glycan-dependent intercellular communication are largely unknown. In the nervous system, glycans play important roles in the development and regulation of synapses mediating intercellular communication. The Drosophila neuromuscular junction serves as a genetically tractable synapse in which expression of glycan-related genes can be systematically knocked down to investigate effects on synaptic morphology and function. This study employs a transgenic RNAi screen to characterize the synaptic requirements of 130 glycan-related genes. From this screen, two functionally paired genes (hs6st and sulf1) that add or remove a sulfate at the 6-O position on heparan sulfate proteoglycans (HSPGs) were identified as being critically important for synaptic functional development. Removal of each gene produces an opposite effect on neurotransmission strength, weakening and strengthening communication, respectively. This mechanism controls the synaptic expression of two HSPGs, which act as co-receptors to control the abundance of anterograde WNT and retrograde BMP signals, which drive intracellular signal transduction pathways regulating gene transcription to control synaptic functional development. This screen serves as a platform for systematic investigation of glycan mechanisms regulating synaptic development.
For nearly 150 years, it has been recognized that cell shape strongly influences the orientation of the mitotic cleavage plane (e.g. Hofmeister, 1863). However, we still understand little about the complex interplay between cell shape and cleavage plane orientation in epithelia, where polygonal cell geometries emerge from multiple factors, including cell packing, cell growth, and cell division itself. Here, using mechanical simulations, we show that the polygonal shapes of individual cells can systematically bias the long axis orientations of their adjacent mitotic neighbors. Strikingly, analysis of both animal epithelia and plant epidermis confirm a robust and nearly identical correlation between local cell topology and cleavage plane orientation in vivo. Using simple mathematics, we show that this effect derives from fundamental packing constraints. Our results suggest that local epithelial topology is a key determinant of cleavage plane orientation, and that cleavage plane bias may be a widespread property of polygonal cell sheets in plants and animals.
The DNA damage checkpoint, the first pathway known to be activated in response to DNA damage, is a mechanism by which the cell cycle is temporarily arrested to allow DNA repair. The checkpoint pathway transmits signals from the sites of DNA damage to the cell cycle machinery through the evolutionarily conserved ATM (ataxia telangiectasia mutated) and ATR (ATM- and Rad3-related) kinase cascades. We conducted a genome-wide RNAi (RNA interference) screen in Drosophila cells to identify previously unknown genes and pathways required for the G2-M checkpoint induced by DNA double-strand breaks (DSBs). Our large-scale analysis provided a systems-level view of the G2-M checkpoint and revealed the coordinated actions of particular classes of proteins, which include those involved in DNA repair, DNA replication, cell cycle control, chromatin regulation, and RNA processing. Further, from the screen and in vivo analysis, we identified previously unrecognized roles of two DNA damage response genes, mus101 and mus312. Our results suggest that the DNA replication preinitiation complex, which includes MUS101, and the MUS312-containing nuclease complexes, which are important for DSB repair, also function in the G2-M checkpoint. Our results provide insight into the diverse mechanisms that link DNA damage and the checkpoint signaling pathway.
Existing transgenic RNAi resources in Drosophila melanogaster based on long double-stranded hairpin RNAs are powerful tools for functional studies, but they are ineffective in gene knockdown during oogenesis, an important model system for the study of many biological questions. We show that shRNAs, modeled on an endogenous microRNA, are extremely effective at silencing gene expression during oogenesis. We also describe our progress toward building a genome-wide shRNA resource.
Characterizing the extent and logic of signaling networks is essential to understanding specificity in such physiological and pathophysiological contexts as cell fate decisions and mechanisms of oncogenesis and resistance to chemotherapy. Cell-based RNA interference (RNAi) screens enable the inference of large numbers of genes that regulate signaling pathways, but these screens cannot provide network structure directly. We describe an integrated network around the canonical receptor tyrosine kinase (RTK)–Ras–extracellular signal–regulated kinase (ERK) signaling pathway, generated by combining parallel genome-wide RNAi screens with protein-protein interaction (PPI) mapping by tandem affinity purification–mass spectrometry. We found that only a small fraction of the total number of PPI or RNAi screen hits was isolated under all conditions tested and that most of these represented the known canonical pathway components, suggesting that much of the core canonical ERK pathway is known. Because most of the newly identified regulators are likely cell type– and RTK-specific, our analysis provides a resource for understanding how output through this clinically relevant pathway is regulated in different contexts. We report in vivo roles for several of the previously unknown regulators, including CG10289 and PpV, the Drosophila orthologs of two components of the serine/threonine–protein phosphatase 6 complex; the Drosophila ortholog of TepIV, a glycophosphatidylinositol-linked protein mutated in human cancers; CG6453, a noncatalytic subunit of glucosidase II; and Rtf1, a histone methyltransferase.
An important step in epithelial organ development is size maturation of the organ lumen to attain correct dimensions. Here we show that the regulated expression of Tenectin (Tnc) is critical to shape the Drosophila melanogaster hindgut tube. Tnc is a secreted protein that fills the embryonic hindgut lumen during tube diameter expansion. Inside the lumen, Tnc contributes to detectable O-Glycans and forms a dense striated matrix. Loss of tnc causes a narrow hindgut tube, while Tnc over-expression drives tube dilation in a dose-dependent manner. Cellular analyses show that luminal accumulation of Tnc causes an increase in inner and outer tube diameter, and cell flattening within the tube wall, similar to the effects of a hydrostatic pressure in other systems. When Tnc expression is induced only in cells at one side of the tube wall, Tnc fills the lumen and equally affects all cells at the lumen perimeter, arguing that Tnc acts non-cell-autonomously. Moreover, when Tnc expression is directed to a segment of a tube, its luminal accumulation is restricted to this segment and affects the surrounding cells to promote a corresponding local diameter expansion. These findings suggest that deposition of Tnc into the lumen might contribute to expansion of the lumen volume, and thereby to stretching of the tube wall. Consistent with such an idea, ectopic expression of Tnc in different developing epithelial tubes is sufficient to cause dilation, while epidermal Tnc expression has no effect on morphology. Together, the results show that epithelial tube diameter can be modelled by regulating the levels and pattern of expression of a single luminal glycoprotein.
Epithelial tubes constitute the functional units of vital organs, and they undergo highly regulated changes in size and shape during development to accommodate the three-dimensional configurations optimal for organ physiology. Through studies of Drosophila melanogaster, we show that epithelial tube diameter can be modelled simply by regulating the levels and pattern of expression of a single glycoprotein. The protein is secreted into the tubular lumen, where it forms a dense matrix and acts in a dose-dependent manner to drive diameter growth. We suggest that deposition of the protein into the lumen promotes local expansion of the lumen volume, and thereby stretching of the surrounding tube wall. Such a mechanism could represent a general means to adjust tube diameter during epithelial organ development.
RNA interference (RNAi) provides a powerful reverse genetics approach to analyze gene functions both in tissue culture and in vivo. Because of its widespread applicability and effectiveness it has become an essential part of the tool box kits of model organisms such as Caenorhabditis elegans, Drosophila, and the mouse. In addition, the use of RNAi in animals in which genetic tools are either poorly developed or nonexistent enables a myriad of fundamental questions to be asked. Here, we review the methods and applications of in vivo RNAi to characterize gene functions in model organisms and discuss their impact to the study of developmental as well as evolutionary questions. Further, we discuss the applications of RNAi technologies to crop improvement, pest control and RNAi therapeutics, thus providing an appreciation of the potential for phenomenal applications of RNAi to agriculture and medicine.
In vivo RNAi technology is replacing classical genetics in screens and conditional targeting of gene function. It also has applications in crop improvement, pest control, and medicine.
In Drosophila melanogaster, cis-regulatory modules that are activated by the Notch cell–cell signaling pathway all contain two types of transcription factor binding sites: those for the pathway's transducing factor Suppressor of Hairless [Su(H)] and those for one or more tissue- or cell type–specific factors called “local activators.” The use of different “Su(H) plus local activator” motif combinations, or codes, is critical to ensure that only the correct subset of the broadly utilized Notch pathway's target genes are activated in each developmental context. However, much less is known about the role of enhancer “architecture”—the number, order, spacing, and orientation of its component transcription factor binding motifs—in determining the module's specificity. Here we investigate the relationship between architecture and function for two Notch-regulated enhancers with spatially distinct activities, each of which includes five high-affinity Su(H) sites. We find that the first, which is active specifically in the socket cells of external sensory organs, is largely resistant to perturbations of its architecture. By contrast, the second enhancer, active in the “non-SOP” cells of the proneural clusters from which neural precursors arise, is sensitive to even simple rearrangements of its transcription factor binding sites, responding with both loss of normal specificity and striking ectopic activity. Thus, diverse cryptic specificities can be inherent in an enhancer's particular combination of transcription factor binding motifs. We propose that for certain types of enhancer, architecture plays an essential role in determining specificity, not only by permitting factor–factor synergies necessary to generate the desired activity, but also by preventing other activator synergies that would otherwise lead to unwanted specificities.
Enhancers, or cis-regulatory modules, are the major transcriptional control elements in the genome. Some enhancers are activated by the Notch cell–cell signaling pathway, in many different cellular contexts in the same organism. It is well established that the cell type–specific activity of each such enhancer depends in part on its particular combination of transcription factor binding sites. But how important is the enhancer's “architecture”—the number, order, spacing, and orientation of these binding sites—in determining its function and specificity? Here we have examined in detail the role of architecture in the activity of two different Notch-regulated enhancers. We find that one is largely insensitive to rearrangement of its important factor binding sites. But even simple alterations can cause the second enhancer to lose much of its normal activity and to become active in novel territories. By more substantial rearrangements, we can even convert the specificity of the second enhancer to that of the first. Therefore, besides binding site composition, the architecture of some enhancers also plays an essential role in determining when and where they respond to the same signaling pathway during development.
CBP and the related p300 protein are widely used transcriptional co-activators in metazoans that interact with multiple transcription factors. Whether CBP/p300 occupies the genome equally with all factors or preferentially binds together with some factors is not known. We therefore compared Drosophila melanogaster CBP (nejire) ChIP–seq peaks with regions bound by 40 different transcription factors in early embryos, and we found high co-occupancy with the Rel-family protein Dorsal. Dorsal is required for CBP occupancy in the embryo, but only at regions where few other factors are present. CBP peaks in mutant embryos lacking nuclear Dorsal are best correlated with TGF-ß/Dpp-signaling and Smad-protein binding. Differences in CBP occupancy in mutant embryos reflect gene expression changes genome-wide, but CBP also occupies some non-expressed genes. The presence of CBP at silent genes does not result in histone acetylation. We find that Polycomb-repressed H3K27me3 chromatin does not preclude CBP binding, but restricts histone acetylation at CBP-bound genomic sites. We conclude that CBP occupancy in Drosophila embryos preferentially overlaps factors controlling dorso-ventral patterning and that CBP binds silent genes without causing histone hyperacetylation.
Development of an embryo into different cell types relies on regulation of gene expression, whereby genes are coordinately turned on or off. CBP and the related p300 protein are central regulators of gene expression in animal cells. These co-activator proteins facilitate gene activation by a multitude of transcription factors, possibly through their histone acetyltransferase activity. Consequently, loss of CBP/p300 gene function disrupts development and is lethal in mice, worms, and flies. How CBP/p300 is targeted to regulatory DNA sequences is not understood. Here, we have compared genome occupancy of CBP with 40 different transcription factors in Drosophila embryos and find that master regulators of dorsal-ventral patterning, the transcription factors Dorsal and Medea, target CBP to the genome. CBP occupies mainly active genes in embryos, where histones become acetylated. Surprisingly, the presence of CBP at silent genes does not result in histone acetylation. We find that repressive chromatin prevents histone acetylation by CBP. Our results demonstrate that CBP preferentially associates with some gene regulatory networks and that CBP binds silent genes without causing histone acetylation. These data have implications for prediction of transcriptional regulatory sequences and for understanding gene regulation by one of the most widely used co-activators in animal cells.
The homeotic genes in Drosophila melanogaster are aligned on the chromosome in the order of the body segments that they affect. The genes affecting the more posterior segments repress the more anterior genes. This posterior dominance rule must be qualified in the case of abdominal-A (abd-A) repression by Abdominal-B (Abd-B). Animals lacking Abd-B show ectopic expression of abd-A in the epidermis of the eighth abdominal segment, but not in the central nervous system. Repression in these neuronal cells is accomplished by a 92 kb noncoding RNA. This “iab-8 RNA” produces a micro RNA to repress abd-A, but also has a second, redundant repression mechanism that acts only “in cis.” Transcriptional interference with the abd-A promoter is the most likely mechanism.
Although long, noncoding RNAs have been found in many organisms, it has been difficult to assign to them any molecular function. The homeotic gene clusters in the fruit fly, Drosophila melanogaster, contain many such noncoding RNAs. We have characterized one such noncoding RNA, a 92 kb transcription unit from within the bithorax complex. This transcript, called the iab-8 ncRNA, is made in the cells of the central nervous system in the eighth abdominal segment, along with the homeotic transcription factor Abdominal-B. Another homeotic transcription factor, abdominal-A, is repressed in these cells. It has generally been assumed that abdominal-A repression in these cells is mediated by the Abdominal-B protein. However, here we show that it is not Abdominal-B that represses abdominal-A, but the iab-8 ncRNA. This repression is accomplished by two redundant mechanisms; the iab-8 precursor produces a micro RNA, which targets the abdominal-A mRNA, and iab-8 transcription interferes with the abdominal-A promoter, which lies just downstream of the iab-8 ncRNA poly(A) site.
Drosophila melanogaster Held Out Wings (HOW) is a conserved RNA–binding protein (RBP) belonging to the STAR family, whose closest mammalian ortholog Quaking (QKI) has been implicated in embryonic development and nervous system myelination. The HOW RBP modulates a variety of developmental processes by controlling mRNA levels and the splicing profile of multiple key regulatory genes; however, mechanisms regulating its activity in tissues have yet to be elucidated. Here, we link receptor tyrosine kinase (RTK) signaling to the regulation of QKI subfamily of STAR proteins, by showing that HOW undergoes phosphorylation by MAPK/ERK. Importantly, we show that this modification facilitates HOW dimerization and potentiates its ability to bind RNA and regulate its levels. Employing an antibody that specifically recognizes phosphorylated HOW, we show that HOW is phosphorylated in embryonic muscles and heart cardioblasts in vivo, thus documenting for the first time Serine/Threonine (Ser/Thr) phosphorylation of a STAR protein in the context of an intact organism. We also identify the sallimus/D-titin (sls) gene as a novel muscle target of HOW–mediated negative regulation and further show that this regulation is phosphorylation-dependent, underscoring the physiological relevance of this modification. Importantly, we demonstrate that HOW Thr phosphorylation is reduced following muscle-specific knock down of Drosophila MAPK rolled and that, correspondingly, Sls is elevated in these muscles, similarly to the HOW RNAi effect. Taken together, our results provide a coherent mechanism of differential HOW activation; MAPK/ERK-dependent phosphorylation of HOW promotes the formation of HOW dimers and thus enhances its activity in controlling mRNA levels of key muscle-specific genes. Hence, our findings bridge between MAPK/ERK signaling and RNA regulation in developing muscles.
Somatic muscles are huge cells that feature highly organized sarcomeric architecture, whose formation and maintenance are not fully understood. Multiple signals play a role in these cells, including the highly conserved MAPK/ERK pathway, which often serves as a cue for cellular proliferation or differentiation. In this study, we reveal a role for MAPK/ERK signaling in the regulation of multiple muscle genes at the level of mRNA, through the control of the activity of the Drosophila RNA–binding protein Held Out wings (HOW). Specifically, we show that HOW undergoes phosphorylation by MAPK/ERK, which increases its ability to form dimers and enhances its RNA–binding capacity. We further demonstrate that HOW is phosphorylated in embryonic and larval muscles by MAPK in vivo and that this event is important for its ability to regulate the levels of a giant sarcomeric gene homologous to vertebrate titin, thus contributing to the maintenance of muscle sarcomeric architecture. Importantly, HOW is a close homolog of mammalian Quaking, an essential protein in embryonic development and nervous system myelination, a reduction of which is correlated with Schizophrenia. Thus, our results raise the possibility that MAPK/ERK phosphorylation similarly regulates RNA profiles in other tissues controlled by proteins of the conserved Quaking family.
RNAi Screens in Drosophila and human cells for novel actin regulators revealed conserved roles for proteins involved in nuclear actin export, RNA splicing, and ubiquitination.
Although a large number of actin-binding proteins and their regulators have been identified through classical approaches, gaps in our knowledge remain. Here, we used genome-wide RNA interference as a systematic method to define metazoan actin regulators based on visual phenotype. Using comparative screens in cultured Drosophila and human cells, we generated phenotypic profiles for annotated actin regulators together with proteins bearing predicted actin-binding domains. These phenotypic clusters for the known metazoan “actinome” were used to identify putative new core actin regulators, together with a number of genes with conserved but poorly studied roles in the regulation of the actin cytoskeleton, several of which we studied in detail. This work suggests that although our search for new components of the core actin machinery is nearing saturation, regulation at the level of nuclear actin export, RNA splicing, ubiquitination, and other upstream processes remains an important but unexplored frontier of actin biology.
Proper assignment of cellular fates relies on correct interpretation of Wnt and Hedgehog (Hh) signals. Members of the Wnt Inhibitory Factor-1 (WIF1) family are secreted modulators of these extracellular signaling pathways. Vertebrate WIF1 binds Wnts and inhibits their signaling, but its Drosophila melanogaster ortholog Shifted (Shf) binds Hh and extends the range of Hh activity in the developing D. melanogaster wing. Shf activity is thought to depend on reinforcing interactions between Hh and glypican HSPGs. Using zebrafish embryos and the heterologous system provided by D. melanogaster wing, we report on the contribution of glypican HSPGs to the Wnt-inhibiting activity of zebrafish Wif1 and on the protein domains responsible for the differences in Wif1 and Shf specificity. We show that Wif1 strengthens interactions between Wnt and glypicans, modulating the biphasic action of glypicans towards Wnt inhibition; conversely, glypicans and the glypican-binding “EGF-like” domains of Wif1 are required for Wif1's full Wnt-inhibiting activity. Chimeric constructs between Wif1 and Shf were used to investigate their specificities for Wnt and Hh signaling. Full Wnt inhibition required the “WIF” domain of Wif1, and the HSPG-binding EGF-like domains of either Wif1 or Shf. Full promotion of Hh signaling requires both the EGF-like domains of Shf and the WIF domains of either Wif1 or Shf. That the Wif1 WIF domain can increase the Hh promoting activity of Shf's EGF domains suggests it is capable of interacting with Hh. In fact, full-length Wif1 affected distribution and signaling of Hh in D. melanogaster, albeit weakly, suggesting a possible role for Wif1 as a modulator of vertebrate Hh signaling.
In developing organisms, cells choose between alternative fates in order to make appropriately patterned tissues, and misregulation of those choices can underlie both developmental defects and cancers. Cells often make these decisions because of signals received from neighboring cells, such as those mediated by the secreted signaling proteins of the Wnt and Hedgehog (Hh) families. While signaling can be regulated by the levels of signaling or receptor proteins expressed by cells, another level of control is exerted by proteins that bind signaling proteins outside of cells and either inhibit or promote the signaling process. In the fruitfly Drosophila
melanogaster, the secreted Shifted protein has been shown to bind Hh and to increase Hh signaling, likely by reinforcing interactions between Hh and cell surface proteins of the glypican family. We provide evidence that the vertebrate homolog of Shifted, Wnt Inhibitory Factor-1 (Wif1), inhibits Wnt activity by a similar mechanism, reinforcing interactions between Wnts and glypicans in a manner that sequesters Wnts from their receptors. We also examine the structural basis for the specificities of Wif1 and Shifted for Wnt and Hh signaling, respectively, and provide evidence that Wif1, although a potent inhibitor of Wnt activity, influences D. melanogaster Hh signaling.
The major facilitator superfamily (MFS) transporter Pho84 and the type III transporter Pho89 are responsible for metabolic effects of inorganic phosphate in yeast. While the Pho89 ortholog Pit1 was also shown to be involved in phosphate-activated MAPK in mammalian cells, it is currently unknown, whether orthologs of Pho84 have a role in phosphate-sensing in metazoan species. We show here that the activation of MAPK by phosphate observed in mammals is conserved in Drosophila cells, and used this assay to characterize the roles of putative phosphate transporters. Surprisingly, while we found that RNAi-mediated knockdown of the fly Pho89 ortholog dPit had little effect on the activation of MAPK in Drosophila S2R+ cells by phosphate, two Pho84/SLC17A1–9 MFS orthologs (MFS10 and MFS13) specifically inhibited this response. Further, using a Xenopus oocyte assay, we show that MSF13 mediates uptake of [33P]-orthophosphate in a sodium-dependent fashion. Consistent with a role in phosphate physiology, MSF13 is expressed highest in the Drosophila crop, midgut, Malpighian tubule, and hindgut. Altogether, our findings provide the first evidence that Pho84 orthologs mediate cellular effects of phosphate in metazoan cells. Finally, while phosphate is essential for Drosophila larval development, loss of MFS13 activity is compatible with viability indicating redundancy at the levels of the transporters.
Polycomb group (PcG) proteins are conserved chromatin factors that maintain silencing of key developmental genes outside of their expression domains. Recent genome-wide analyses showed a Polycomb (PC) distribution with binding to discrete PcG response elements (PREs). Within the cell nucleus, PcG proteins localize in structures called PC bodies that contain PcG-silenced genes, and it has been recently shown that PREs form local and long-range spatial networks. Here, we studied the nuclear distribution of two PcG proteins, PC and Polyhomeotic (PH). Thanks to a combination of immunostaining, immuno-FISH, and live imaging of GFP fusion proteins, we could analyze the formation and the mobility of PC bodies during fly embryogenesis as well as compare their behavior to that of the condensed fraction of euchromatin. Immuno-FISH experiments show that PC bodies mainly correspond to 3D structural counterparts of the linear genomic domains identified in genome-wide studies. During early embryogenesis, PC and PH progressively accumulate within PC bodies, which form nuclear structures localized on distinct euchromatin domains containing histone H3 tri-methylated on K27. Time-lapse analysis indicates that two types of motion influence the displacement of PC bodies and chromatin domains containing H2Av-GFP. First, chromatin domains and PC bodies coordinately undergo long-range motions that may correspond to the movement of whole chromosome territories. Second, each PC body and chromatin domain has its own fast and highly constrained motion. In this motion regime, PC bodies move within volumes slightly larger than those of condensed chromatin domains. Moreover, both types of domains move within volumes much smaller than chromosome territories, strongly restricting their possibility of interaction with other nuclear structures. The fast motion of PC bodies and chromatin domains observed during early embryogenesis strongly decreases in late developmental stages, indicating a possible contribution of chromatin dynamics in the maintenance of stable gene silencing.
The three-dimensional organization of genes and associated proteins is critical for gene regulation. Polycomb group proteins are important developmental regulators controlling the expression of hundreds of genes. They are not homogeneously distributed in the cell nucleus, instead forming nuclear subcompartments called Polycomb bodies. We investigated the dynamics of Polycomb bodies during Drosophila embryonic development, demonstrating that two Polycomb proteins, Polycomb and Polyhomeotic, gradually assemble onto bodies enriched in histone H3 trimethylated on lysine 27, a hallmark of Polycomb silencing. Polycomb bodies are not the most condensed euchromatic part of the genome. Instead, a large amount of genomic chromatin is organized in a histone- and DNA–dense structure distinct from Polycomb bodies. Polycomb bodies move, meet, and split dynamically during development. Their motion has two regimes: a fast, highly constrained motion and a slower regime where multiple bodies undergo long-range coordinated movements potentially corresponding to chromosome territory movements. These regimes are not restricted to Polycomb but also extend to bulk “condensed euchromatin,” which is characterized by slower motion and a narrower radius of confinement. Both motion regimes progressively slow down during development, suggesting that regulation of chromatin dynamics may play an important role in the maintenance of gene silencing in differentiated cells.
Epigenetic regulation plays critical roles in the regulation of cell proliferation, fate determination, and survival. It has been shown to control self-renewal and lineage differentiation of embryonic stem cells. However, epigenetic regulation of adult stem cell function remains poorly defined. Drosophila ovarian germline stem cells (GSCs) are a productive adult stem cell system for revealing regulatory mechanisms controlling self-renewal and differentiation. In this study, we show that Eggless (Egg), a H3K9 methyltransferase in Drosophila, is required in GSCs for controlling self-renewal and in escort cells for regulating germ cell differentiation. egg mutant ovaries primarily exhibit germ cell differentiation defects in young females and gradually lose GSCs with time, indicating that Egg regulates both germ cell maintenance and differentiation. Marked mutant egg GSCs lack expression of trimethylated H3K9 (H3k9me3) and are rapidly lost from the niche, but their mutant progeny can still differentiate into 16-cell cysts, indicating that Egg is required intrinsically to control GSC self-renewal but not differentiation. Interestingly, BMP-mediated transcriptional repression of differentiation factor bam in marked egg mutant GSCs remains normal, indicating that Egg is dispensable for BMP signaling in GSCs. Normally, Bam and Bgcn interact with each other to promote GSC differentiation. Interestingly, marked double mutant egg bgcn GSCs are still lost, but their progeny are able to differentiate into 16-cell cysts though bgcn mutant GSCs normally do not differentiate, indicating that Egg intrinsically controls GSC self-renewal through repressing a Bam/Bgcn-independent pathway. Surprisingly, RNAi-mediated egg knockdown in escort cells leads to their gradual loss and a germ cell differentiation defect. The germ cell differentiation defect is at least in part attributed to an increase in BMP signaling in the germ cell differentiation niche. Therefore, this study has revealed the essential roles of histone H3K9 trimethylation in controlling stem cell maintenance and differentiation through distinct mechanisms.
Epigenetic regulation plays critical roles in the regulation of cell proliferation, fate determination, and survival. It has been extensively studied in embryonic stem cells for its roles in the control of self-renewal and lineage differentiation. However, epigenetic regulation of adult stem cell function remains poorly defined. In this study, we show that Eggless (Egg), a H3K9 methyltransferase in Drosophila, is required in germline stem cells (GSCs) for controlling self-renewal and in escort cells for regulating germ cell differentiation. egg mutant ovaries exhibit both germ cell differentiation defects and GSC loss, indicating that Egg regulates both germ cell maintenance and differentiation. Intrinsic inactivation of egg function in GSCs leads to loss of trimethylated H3K9 expression and rapid departure from the niche, indicating that Egg is required intrinsically to control GSC self-renewal. Our genetic results reveal that Egg intrinsically controls GSC self-renewal through repressing a Bam/Bgcn-independent pathway. Furthermore, RNAi-mediated egg knockdown in escort cells leads to germ cell differentiation defects due to increased BMP signaling. Therefore, this study has revealed essential roles of histone H3K9 trimethylation in controlling stem cell maintenance and differentiation through distinct mechanisms in the Drosophila ovary.
The ribosome is critical for all aspects of cell growth due to its essential role in protein synthesis. Paradoxically, many Ribosomal proteins (Rps) act as tumour suppressors in Drosophila and vertebrates. To examine how reductions in Rps could lead to tissue overgrowth, we took advantage of the observation that an RpS6 mutant dominantly suppresses the small rough eye phenotype in a cyclin E hypomorphic mutant (cycEJP). We demonstrated that the suppression of cycEJP by the RpS6 mutant is not a consequence of restoring CycE protein levels or activity in the eye imaginal tissue. Rather, the use of UAS-RpS6 RNAi transgenics revealed that the suppression of cycEJP is exerted via a mechanism extrinsic to the eye, whereby reduced Rp levels in the prothoracic gland decreases the activity of ecdysone, the steroid hormone, delaying developmental timing and hence allowing time for tissue and organ overgrowth. These data provide for the first time a rationale to explain the counter-intuitive organ overgrowth phenotypes observed for certain members of the Minute class of Drosophila Rp mutants. They also demonstrate how Rp mutants can affect growth and development cell non-autonomously.
Ribosomes are required for protein synthesis, which is essential for cell growth and division, thus mutations that reduce Rp expression would be expected to limit cell growth. Paradoxically, heterozygous deletion or mutation of certain Rps can actually promote growth and proliferation and in some cases bestow predisposition to cancer. The underlying mechanism(s) behind these unexpected overgrowth phenotypes despite impairment of ribosome biogenesis has remained obscure. We have addressed this question using the power of Drosophila genetics, taking advantage of our observation that four different Rp mutants, or Minutes, are able to suppress a small rough eye phenotype associated with a mutation of the essential controller of cell proliferation cyclin E (cycEJP). Our findings demonstrate that suppression of cycEJP by the RpS6 mutant is exerted via a tissue non-autonomous mechanism whereby reduced Rp in the prothoracic gland decreases activity of the steroid hormone ecdysone, delaying development and hence allowing time for compensatory growth. These data provide for the first time a rationale to explain the counter-intuitive organ overgrowth phenotypes observed for certain Drosophila Minutes. Our findings also have implications for the effect of Rp mutants on endocrine related control of tissue growth in higher organisms.
FlyRNAi (http://www.flyrnai.org), the database and website of the Drosophila RNAi Screening Center (DRSC) at Harvard Medical School, serves a dual role, tracking both production of reagents for RNA interference (RNAi) screening in Drosophila cells and RNAi screen results. The database and website is used as a platform for community availability of protocols, tools, and other resources useful to researchers planning, conducting, analyzing or interpreting the results of Drosophila RNAi screens. Based on our own experience and user feedback, we have made several changes. Specifically, we have restructured the database to accommodate new types of reagents; added information about new RNAi libraries and other reagents; updated the user interface and website; and added new tools of use to the Drosophila community and others. Overall, the result is a more useful, flexible and comprehensive website and database.