Michael Boutros

REST: A mammalian silencer protein that restricts sodium channel gene expression to neurons

Expression of the type II voltage-dependent sodium channel gene is restricted to neurons by a silencer element active in nonneuronal cells. We have cloned cDNA coding for a transcription factor (REST) that binds to this silencer element. Expression of a recombinant REST protein confers the ability to silence type II reporter genes in neuronal cell types lacking the native REST protein, whereas expression of a dominant negative form of REST in nonneuronal cells relieves silencing mediated by the native protein. REST transcripts in developing mouse embryos are detected ubiquitously outside of the nervous system. We propose that expression of the type II sodium channel gene in neurons reflects a default pathway that is blocked in nonneuronal cells by the presence of REST.

Dishevelled Activates JNK and Discriminates between JNK Pathways in Planar Polarity and wingless Signaling

Frizzled family proteins have been described as receptors of Wnt signaling molecules. In Drosophila, the two known Frizzled proteins are associated with distinct developmental processes. Genesis of epithelial planar polarity requires Frizzled, whereas Dfz2 affects morphogenesis by wingless-mediated signaling. Dishevelled is required in both signaling pathways. Here, we use genetic and overexpression assays to show that Dishevelled activates JNK cascades. Rescue analysis reveals different protein domain requirements in Dishevelled for the two pathways; the C-terminal DEP domain is essential to rescue planar polarity defects and induce JNK signaling. Furthermore, the planar polarity-specific dsh1 allele is mutated in the DEP domain. Our results indicate that different Wnt/Fz signals activate distinct intracellular pathways, and Dishevelled discriminates among them by distinct domain interactions.

Sequential Activation of Signaling Pathways during Innate Immune Responses in Drosophila

Innate immunity is essential for metazoans to fight microbial infections. Genome-wide expression profiling was used to analyze the outcome of impairing specific signaling pathways after microbial challenge. We found that these transcriptional patterns can be dissected into distinct groups. We demonstrate that, in addition to signaling through the Toll and Imd pathways, signaling through the JNK and JAK/STAT pathways controls distinct subsets of targets induced by microbial agents. Each pathway shows a specific temporal pattern of activation and targets different functional groups, suggesting that innate immune responses are modular and recruit distinct physiological programs. In particular, our results may imply a close link between the control of tissue repair and antimicrobial processes.

Secretion of Wnt Ligands Requires Evi, a Conserved Transmembrane Protein

Wnt signaling pathways are important for multiple biological processes during development and disease. Wnt proteins are secreted factors that activate target-gene expression in both a short- and long-range manner. Currently, little is known about how Wnts are released from cells and which factors facilitate their secretion. Here, we identify a conserved multipass transmembrane protein, Evenness interrupted (Evi/Wls), through an RNAi survey for transmembrane proteins involved in Drosophila Wingless (Wg) signaling. During development, evi mutants have patterning defects that phenocopy wg loss-of-function alleles and fail to express Wg target genes. evis function is evolutionarily conserved as depletion of its human homolog disrupts Wnt signaling in human cells. Epistasis experiments and clonal analysis place evi in the Wg-producing cell. Our results show that Wg is retained by evi mutant cells and suggest that evi is the founding member of a gene family specifically required for Wg/Wnt secretion.

Active Wnt proteins are secreted on exosomes

Wnt signalling has important roles during development and in many diseases. As morphogens, hydrophobic Wnt proteins exert their function over a distance to induce patterning and cell differentiation decisions. Recent studies have identified several factors that are required for the secretion of Wnt proteins; however, how Wnts travel in the extracellular space remains a largely unresolved question. Here we show that Wnts are secreted on exosomes both during Drosophila development and in human cells. We demonstrate that exosomes carry Wnts on their surface to induce Wnt signalling activity in target cells. Together with the cargo receptor Evi/WIs, Wnts are transported through endosomal compartments onto exosomes, a process that requires the R-SNARE Ykt6. Our study demonstrates an evolutionarily conserved functional role of extracellular vesicular transport of Wnt proteins.

Requirement of Prorenin Receptor and Vacuolar H + -ATPase-Mediated Acidification for Wnt Signaling

Of Wnt, Prorenin Receptor, and V-ATPase The Wnt protein binds to receptors at the cell surface and regulates signaling pathways that control a wide range of critical biological processes from stem cell differentiation to generation of cancer. In a screen for components required for Wnt signaling, Cruciat et al. (p. 459) discovered an unexpected partner, the prorenin receptor (PRR). PRR bound to the Wnt receptor proteins Fz8 and LRP6. The PRR protein could interact with the receptors and promote Wnt signaling without its cytoplasmic domain through which it initiates signaling in response to the prorenin protein. Its role in Wnt signaling appears to be rather different. The PRR binds to the vacuolar H+–adenosine triphosphatase (V-ATPase), a proton pump that can influence endocytosis by acidification of vesicles. The V-ATPase was also necessary for phosphorylation of LRP6 and Wnt. Thus, PRR may link the V-ATPase to the Wnt receptor protein LRP6, allowing acidification in the vicinity of the activated receptor, which appears to be necessary for phosphorylation of LRP6 and subsequent signaling. A dual-function protein acts as an adaptor in a major developmental signaling pathway. Wnt/β-catenin signaling is important in stem cell biology, embryonic development, and disease, including cancer. However, the mechanism of Wnt signal transmission, notably how the receptors are activated, remains incompletely understood. We found that the prorenin receptor (PRR) is a component of the Wnt receptor complex. PRR functions in a renin-independent manner as an adaptor between Wnt receptors and the vacuolar H+–adenosine triphosphatase (V-ATPase) complex. Moreover, PRR and V-ATPase were required to mediate Wnt signaling during antero-posterior patterning of Xenopus early central nervous system development. The results reveal an unsuspected role for the prorenin receptor, V-ATPase activity, and acidification during Wnt/β-catenin signaling.

Signaling Role of Hemocytes in Drosophila JAK/STAT-Dependent Response to Septic Injury

To characterize the features of JAK/STAT signaling in Drosophila immune response, we have identified totA as a gene that is regulated by the JAK/STAT pathway in response to septic injury. We show that septic injury triggers the hemocyte-specific expression of upd3, a gene encoding a novel Upd-like cytokine that is necessary for the JAK/STAT-dependent activation of totA in the Drosophila counterpart of the mammalian liver, the fat body. In addition, we demonstrate that totA activation also requires the NF-KB-like Relish pathway, indicating that fat body cells integrate the activity of NF-KB and JAK/STAT signaling pathways upon immune response. This study reveals that, in addition to the pattern recognition receptor-mediated NF-KB-dependent immune response, Drosophila undergoes a complex systemic response that is mediated by the production of cytokines in blood cells, a process that is similar to the acute phase response in mammals.

LGR4 and LGR5 are R-spondin receptors mediating Wnt/β-catenin and Wnt/PCP signalling

R‐spondins are secreted Wnt signalling agonists, which regulate embryonic patterning and stem cell proliferation, but whose mechanism of action is poorly understood. Here we show that R‐spondins bind to the orphan G‐protein‐coupled receptors LGR4 and LGR5 by their Furin domains. Gain‐ and loss‐of‐function experiments in mammalian cells and Xenopus embryos indicate that LGR4 and LGR5 promote R‐spondin‐mediated Wnt/β‐catenin and Wnt/PCP signalling. R‐spondin‐triggered β‐catenin signalling requires Clathrin, while Wnt3a‐mediated β‐catenin signalling requires Caveolin‐mediated endocytosis, suggesting that internalization has a mechanistic role in R‐spondin signalling.

The art and design of genetic screens: RNA interference

The remarkable gene knockdown technique of RNAi has opened exciting new avenues for genetic screens in model organisms and human cells. Here we describe the current state of the art for RNAi screening, and stress the importance of well-designed assays and of analytical approaches for large-scale screening experiments, from high-throughput screens using simplified homogenous assays to microscopy and whole-animal experiments. Like classical genetic screens in the past, the success of large-scale RNAi surveys depends on a careful development of phenotypic assays and their interpretation in a relevant biological context.

E-CRISP: fast CRISPR target site identification

encodes an RNA (crRNA), consisting of a guide RNA (gRNA) and transactivating CRISPR RNA parts. A processed crRNA fragment is incorporated into the Cas9 protein, guiding it to the target DNA, where the Cas9 nuclease introduces a double-strand break9,10. The CRISPR-Cas system has been successfully used in human induced pluripotent stem cells, mice, zebrafish and flies, among other organisms, to disrupt gene function. Here we describe E-CRISP, a web application to design gRNA sequences (Fig. 1a). It provides flexible output and experimentoriented design parameters, enabling design of multiple libraries and thereby systematic analysis of the influence of different parameters. E-CRISP identifies target sequences complementary to the gRNA ending in a 3ʹ protospacer-adjacent motif (PAM), N(G or A)G, which is required for the recruited Cas9 nuclease to cut the DNA double strand. E-CRISP uses a fast indexing approach to find binding sites and a binary interval tree for rapid annotation of putative gRNA target sites (Supplementary Note 1). Using these algorithms, it is feasible to create genome-scale libraries for several organisms in a few hours. For instance, to design a library covering the Drosophila melanogaster genome requires less than 1 h (Supplementary Fig. 1 and Supplementary Table 1). Off-target effects and target-site homology are evaluated by E-CRISP using the alignment program Bowtie2 (Supplementary Note 2). Designs are shown in the output if the number of offtargets does not exceed a user-specified threshold. If more than one design is found targeting a desired locus, designs are ranked according to on-target specificity and number of off-targets. E-CRISP can also be used to reevaluate CRISPR constructs for onor off-target sites and targeted genomic loci. As an example, we searched for designs to target let-7 for gene disruption in zebrafish, fly, worm and human (Fig. 1b). We found at least one gRNA design per locus. In worm, fly and human, the cuts are located at the site that is transformed to mature microRNA and thus should lead to mutations blocking its proper function. In zebrafish the cut is located in the predicted hairpin structure. E-CRISP is available for twelve organisms and can be easily extended. E-CRISP will help to further develop and deploy the acKnoWLedGments This work was supported by the Wellcome Trust through a Senior Research Fellowship to J.R. (084229), a core grant to the Wellcome Trust Centre for Cell Biology (092076), a European Research Council grant (233457) to M.T., a Genome Québec International Recruitment Award to M.T. and a Canada Research Chair in Systems and Synthetic Biology to M.T.