Efrat Oron

The COP9 signalosome is essential for development of Drosophila melanogaster.

The COP9 signalosome (originally described as the COP9 complex) is an essential multi-subunit repressor of light-regulated development in plants [1] [2]. It has also been identified in mammals, though its role remains obscure [3] [4] [5]. This complex is similar to the regulatory lid of the proteasome and eIF3 [5] [9] [10] [11] [12] and several of its subunits are known to be involved in kinase signaling pathways [4] [6] [7] [8]. No proteins homologous to COP9 signalosome components were identified in the Saccharomyces cerevisiae genome, suggesting that the COP9 signalosome is specific for multi-cellular differentiation [13]. In order to reveal the developmental function of the COP9 signalosome in animals, we have isolated Drosophila melanogaster genes encoding eight subunits of the COP9 signalosome, and have shown by co-immunoprecipitation and gel-filtration analysis that these proteins are components of the Drosophila COP9 signalosome. Yeast two-hybrid assays indicated that several of these proteins interact, some through the PCI domain. Disruption of one of the subunits by either a P-element insertion or deletion of the gene caused lethality at the late larval or pupal stages. This lethality is probably a result of numerous pleiotropic effects. Our results indicate that the COP9 signalosome is conserved in invertebrates and that it has an essential role in animal development.

Drosophila JAB1/CSN5 Acts in Photoreceptor Cells to Induce Glial Cells

Different classes of photoreceptor neurons (R cells) in the Drosophila compound eye form connections in different optic ganglia. The R1-R6 subclass connects to the first optic ganglion, the lamina, and relies upon glial cells as intermediate targets. Conversely, R cells promote glial cell development including migration of glial cells into the target region. Here, we show that the JAB1/CSN5 subunit of the COP9 signalosome complex is expressed in R cells, accumulates in the developing optic lobe neuropil, and through the analysis of a unique set of missense mutations, is required in R cells to induce lamina glial cell migration. In these CSN5 alleles, R1-R6 targeting is disrupted. Genetic analysis of protein null alleles further revealed that the COP9 signalosome is required at an earlier stage of development for R cell differentiation.

Genomic analysis of COP9 signalosome function in Drosophila melanogaster reveals a role in temporal regulation of gene expression

The COP9 signalosome (CSN), an eight‐subunit protein complex, is conserved in all higher eukaryotes. CSN intersects the ubiquitin–proteasome pathway, modulating signaling pathways controlling various aspects of development. We are using Drosophila as a model system to elucidate the function of this important complex. Transcriptome data were generated for four csn mutants, sampled at three developmental time points. Our results are highly reproducible, being confirmed using two different experimental setups that entail different microarrays and different controls. Our results indicate that the CSN acts as a transcriptional repressor during development of Drosophila, resulting in achronic gene expression in the csn mutants. ‘Time shift’ analysis with the publicly available Drosophila transcriptome data indicates that genes repressed by the CSN are normally induced primarily during late embryogenesis or during metamorphosis. These temporal shifts are likely due to the roles of the CSN in regulating transcription factors. A null mutation in CSN subunit 4 and hypomorphic mutations in csn5 lead to more severe defects than seen in the csn5‐null mutants strain, suggesting that CSN5 carries only some of the CSN function.

COP9 signalosome subunit 5 (CSN5/Jab1) regulates the development of the Drosophila immune system: effects on Cactus, Dorsal and hematopoiesis

The COP9 signalosome is a multifunctional regulator essential for Drosophila development. A loss‐of‐function mutant in Drosophila COP9 signalosome subunit 5 (CSN5) develops melanotic bodies, a phenotype common to mutants in immune signaling. csn5null larvae accumulated high levels of Cactus that co‐localizes with Dorsal to the nucleus. However, Dorsal‐dependent transcriptional activity remained repressed in the absence of an inducing signal, despite its nuclear localization. Dorsal activity in mutant larvae and NFκB activity in CSN5 down‐regulated mammalian cells can be induced following activation of the Toll/IL‐1 pathway. csn5null larvae contained more hemocytes than wild‐type (wt) larvae. A large portion of these cells have differentiated to lamellocytes (LM), a hemocyte cell type rarely seen in normal larvae. The results presented here indicate that CSN5 is a negative regulator of Dorsal subcellular localization, and of hemocyte proliferation and differentiation. These results further indicate that nuclear localization of Dorsal can be uncoupled from its activation. Surprisingly, CSN5 is not necessary for immune‐induced degradation of Cactus.

Drosophila COP9 signalosome subunit 7 interacts with multiple genomic loci to regulate development

The COP9 signalosome protein complex has a central role in the regulation of development of multicellular organisms. While the function of this complex in ubiquitin-mediated protein degradation is well established, results over the past few years have hinted that the COP9 signalosome may function more broadly in the regulation of gene expression. Here, using DamID technology, we show that COP9 signalosome subunit 7 functionally associates with a large number of genomic loci in the Drosophila genome, and show that the expression of many genes within these loci is COP9 signalosome-dependent. This association is likely direct as we show CSN7 binds DNA in vitro. The genes targeted by CSN7 are preferentially enriched for transcriptionally active regions of the genome, and are involved in the regulation of distinct gene ontology groupings including imaginal disc development and cell-cycle control. In accord, loss of CSN7 function leads to cell-cycle delay and altered wing development. These results indicate that CSN7, and by extension the entire COP9 signalosome, functions directly in transcriptional control. While the COP9 signalosome protein complex has long been known to regulate protein degradation, here we expand the role of this complex by showing that subunit 7 binds DNA in vitro and functions directly in vivo in transcriptional control of developmentally important pathways that are relevant for human health.

The angular interval between the direction of progression and body orientation in normal, alcohol- and cocaine treated fruit flies.

In this study we characterize the coordination between the direction a fruit-fly walks and the direction it faces, as well as offer a methodology for isolating and validating key variables with which we phenotype fly locomotor behavior. Our fundamental finding is that the angular interval between the direction a fly walks and the direction it faces is actively managed in intact animals and modulated in a patterned way with drugs. This interval is small in intact flies, larger with alcohol and much larger with cocaine. The dynamics of this interval generates six coordinative modes that flow smoothly into each other. Under alcohol and much more so under cocaine, straight path modes dwindle and modes involving rotation proliferate. To obtain these results we perform high content analysis of video-tracked open field locomotor behavior. Presently there is a gap between the quality of descriptions of insect behaviors that unfold in circumscribed situations, and descriptions that unfold in extended time and space. While the first describe the coordination between low-level kinematic variables, the second quantify cumulative measures and subjectively defined behavior patterns. Here we reduce this gap by phenotyping extended locomotor behavior in terms of the coordination between low-level kinematic variables, which we quantify, combining into a single field two disparate fields, that of high content phenotyping and that of locomotor coordination. This will allow the study of the genes/brain/locomotor coordination interface in genetically engineered and pharmacologically manipulated animal models of human diseases.

Generative rules of Drosophila locomotor behavior as a candidate homology across phyla

The discovery of shared behavioral processes across phyla is a significant step in the establishment of a comparative study of behavior. We use immobility as an origin and reference for the measurement of fly locomotor behavior; speed, walking direction and trunk orientation as the degrees of freedom shaping this behavior; and cocaine as the parameter inducing progressive transitions in and out of immobility. We characterize and quantify the generative rules that shape Drosophila locomotor behavior, bringing about a gradual buildup of kinematic degrees of freedom during the transition from immobility to normal behavior, and the opposite narrowing down into immobility. Transitions into immobility unfold via sequential enhancement and then elimination of translation, curvature and finally rotation. Transitions out of immobility unfold by progressive addition of these degrees of freedom in the opposite order. The same generative rules have been found in vertebrate locomotor behavior in several contexts (pharmacological manipulations, ontogeny, social interactions) involving transitions in-and-out of immobility. Recent claims for deep homology between arthropod central complex and vertebrate basal ganglia provide an opportunity to examine whether the rules we report also share common descent. Our approach prompts the discovery of behavioral homologies, contributing to the elusive problem of behavioral evolution.

Time-window analysis of developmental gene expression data with multiple genetic backgrounds

We study gene expression data, derived from developing tissues, under multiple genetic backgrounds (mutations). Motivated by the perceived behavior under these background, our main goals are to explore time windows questions: Find a large set of genes that have a similar behavior in two different genetic backgrounds, under an appropriate time shift. Find a model that approximates the dynamics of a gene network in developing tissues at different continuous time windows. We first explain the biological significance of these problems, and then explore their computational complexity, which ranges from polynomial to NP-hard. We developed algorithms and heuristics for the different problems, and ran those on synthetic and biological data, with very encouraging results.

Shared generative rules of locomotor behavior in arthropods and vertebrates

The discovery of shared behavioral processes across phyla is an essential step in the establishment of a comparative study of behavior. We use immobility as an origin and reference for the measurement of locomotor behavior; speed, direction of walking and direction of facing as the three degrees of freedom shaping fly locomotor behavior; and cocaine as the parameter inducing a progressive transition in and out of immobility. In this way we expose and quantify the generative rules that shape part of fruit fly locomotor behavior, bringing about a gradual buildup of freedom during the transition from immobility to normal behavior and a precisely opposite narrowing down during the transition into immobility. During buildup the fly exhibits enhancement and then reduction to normal values of movement along each degree of freedom: first, body rotation in the horizontal plane, then path curvature and then speed of translation. Transition into immobility unfolds by narrowing down of the repertoire in the opposite sequential order, showing reciprocal relations during both buildup and narrowing down. The same generative rules apply to vertebrate locomotor behavior in a variety of contexts involving transition out and into immobility. Recent claims for deep homology between the arthropod central complex and the vertebrate basal ganglia provide an opportunity to examine whether the generative rules we discovered also share common descent. Neurochemical processes mediating the buildup of locomotor behavior in vertebrates could guide the search for equivalent processes in arthropods. The measurement methodology we use prompts the discovery of candidate behavioral homologies. Significance Statement Do flies and mice share the same behavior? By defining immobility as an intrinsic reference point for locomotor behavior we show that the rules that generate the transition from immobility to full blown normal behavior, and from full blown behavior to immobility are shared by fruit flies and mice. These rules constitute a much desired aim of evolutionary biology: the discovery of behavioral homologies across distant phyla. The methodology we use facilitates the discovery of cross-phyletic behavioral homologies, shedding light on the problem of the evolution of behavior.