Subcellular localisations of the CPTI collection of YFP-tagged proteins in Drosophila embryos.

A key challenge in the post-genomic area is to identify the function of the genes discovered, with many still uncharacterised in all metazoans. A first step is transcription pattern characterisation, for which we now have near whole-genome coverage in Drosophila. However, we have much more limited information about the expression and subcellular localisation of the corresponding proteins. The Cambridge Protein Trap Consortium generated, via piggyBac transposition, over 600 novel YFP-trap proteins tagging just under 400 Drosophila loci. Here, we characterise the subcellular localisations and expression patterns of these insertions, called the CPTI lines, in Drosophila embryos. We have systematically analysed subcellular localisations at cellularisation (stage 5) and recorded expression patterns at stage 5, at mid-embryogenesis (stage 11) and at late embryogenesis (stages 15-17). At stage 5, 31% of the nuclear lines (41) and 26% of the cytoplasmic lines (67) show discrete localisations that provide clues on the function of the protein and markers for organelles or regions, including nucleoli, the nuclear envelope, nuclear speckles, centrosomes, mitochondria, the endoplasmic reticulum, Golgi, lysosomes and peroxisomes. We characterised the membranous/cortical lines (102) throughout stage 5 to 10 during epithelial morphogenesis, documenting their apico-basal position and identifying those secreted in the extracellular space. We identified the tricellular vertices as a specialized membrane domain marked by the integral membrane protein Sidekick. Finally, we categorised the localisation of the membranous/cortical proteins during cytokinesis.This is the final version. It was first published by The Company of Biologists in Development at http://dev.biologists.org/content/141/20/4006.long

The Toll-dorsal pathway is required for resistance to viral oral infection in Drosophila

Pathogen entry route can have a strong impact on the result of microbial infections in different hosts, including insects. Drosophila melanogaster has been a successful model system to study the immune response to systemic viral infection. Here we investigate the role of the Toll pathway in resistance to oral viral infection in D. melanogaster. We show that several Toll pathway components, including Spätzle, Toll, Pelle and the NF-kB-like transcription factor Dorsal, are required to resist oral infection with Drosophila C virus. Furthermore, in the fat body Dorsal is translocated from the cytoplasm to the nucleus and a Toll pathway target gene reporter is upregulated in response to Drosophila C Virus infection. This pathway also mediates resistance to several other RNA viruses (Cricket paralysis virus, Flock House virus, and Nora virus). Compared with control, viral titres are highly increased in Toll pathway mutants. The role of the Toll pathway in resistance to viruses in D. melanogaster is restricted to oral infection since we do not observe a phenotype associated with systemic infection. We also show that Wolbachia and other Drosophila-associated microbiota do not interact with the Toll pathway-mediated resistance to oral infection. We therefore identify the Toll pathway as a new general inducible pathway that mediates strong resistance to viruses with a route-specific role. These results contribute to a better understanding of viral oral infection resistance in insects, which is particularly relevant in the context of transmission of arboviruses by insect vectors.Biotechnology and Biological Sciences Research Council (UK) grant BB/E005470/1, Fundação para a Ciência e Tecnologia fellowships: SFRH/BPD/65985/2009, SFRH/BD/51881/2012, SFRH/51885/2012

Within-day variability in microbial concentrations at a UK designated bathing water:Implications for regulatory monitoring and the application of predictive modelling based on historical compliance data

Prediction of bathing water quality is recommended by the World Health Organization (WHO), the European Union (EU) and the United States Environmental Protection Agency (USEPA) and is an established element in bathing water management designed to protect public health. Most commonly, historical regulatory compliance data are used for model calibration and provide the dependent variable for modelling. Independent (or predictor) variables (e.g. rainfall, river flow and received irradiance) measured over some antecedent period are used to deliver prediction of the faecal indicator concentration measured on the day of the regulatory sample collection. The implied linked assumptions of this approach are, therefore, that; (i) the independent variables accurately predict the bathing-day water quality; which is (ii) accurately characterized by the single regulatory sample. Assumption (ii) will not be the case where significant within-day variability in water quality is evident. This study built a detailed record of water quality change through 60 days at a UK coastal bathing water in 2011 using half-hourly samples each subjected to triplicate filtration designed to enhance enumeration precision. On average, the mean daily variation in FIO concentrations exceeded 1 log10 order, with the largest daily variations exceeding 2 log10 orders. Significant diurnality was observed at this bathing water, which would determine its EU Directive compliance category if the regulatory samples were collected at the same time each day. A sampling programme of this intensity has not been reported elsewhere to date and, if this pattern is proven to be characteristic of other bathing waters world-wide, it has significance for: (a) the design of regulatory sampling programmes; (b) the use of historical data to assess compliance, which often comprises a single sample taken at the compliance point on a regular, often weekly, basis; and (c) the use of regulatory compliance data to build predictive models of water quality. Keywords: Bathing water variability faecal indicator

Invaginating endoderm pulls on the extending ectoderm

How genetic programs generate cell-intrinsic forces to shape embryos is actively studied, but less so how tissue-scale physical forces impact morphogenesis. Here we address the role of the latter during axis extension, using Drosophila germband extension (GBE) as a model. We found previously that cells elongate in the anteroposterior (AP) axis in the extending germband, suggesting that an extrinsic tensile force contributed to body axis extension. Here we further characterized the AP cell elongation patterns during GBE, by tracking cells and quantifying their apical cell deformation over time. AP cell elongation forms a gradient culminating at the posterior of the embryo, consistent with an AP-oriented tensile force propagating from there. To identify the morphogenetic movements that could be the source of this extrinsic force, we mapped gastrulation movements temporally using light sheet microscopy to image whole Drosophila embryos. We found that both mesoderm and endoderm invaginations are synchronous with the onset of GBE. The AP cell elongation gradient remains when mesoderm invagination is blocked but is abolished in the absence of endoderm invagination. This suggested that endoderm invagination is the source of the tensile force. We next looked for evidence of this force in a simplified system without polarized cell intercalation, in acellular embryos. Using Particle Image Velocimetry, we identify posteriorwards Myosin II flows towards the presumptive posterior endoderm, which still undergoes apical constriction in acellular embryos as in wildtype. We probed this posterior region using laser ablation and showed that tension is increased in the AP orientation, compared to dorsoventral orientation or to either orientations more anteriorly in the embryo. We propose that apical constriction leading to endoderm invagination is the source of the extrinsic force contributing to germband extension. This highlights the importance of physical interactions between tissues during morphogenesis.This work was funded by a Biotechnology and Biological Sciences Research Council grant BB/ J010278/1 to GBB, RJA and BS, a Wellcome Trust Investigator Award 099234/Z/12/Z to BS and a Herchel Smith Postdoctoral Fellowship from the University of Cambridge to C

Analysis of the expression patterns, subcellular localisations and interaction partners of Drosophila proteins using a pigP protein trap library.

Although we now have a wealth of information on the transcription patterns of all the genes in the Drosophila genome, much less is known about the properties of the encoded proteins. To provide information on the expression patterns and subcellular localisations of many proteins in parallel, we have performed a large-scale protein trap screen using a hybrid piggyBac vector carrying an artificial exon encoding yellow fluorescent protein (YFP) and protein affinity tags. From screening 41 million embryos, we recovered 616 verified independent YFP-positive lines representing protein traps in 374 genes, two-thirds of which had not been tagged in previous P element protein trap screens. Over 20 different research groups then characterized the expression patterns of the tagged proteins in a variety of tissues and at several developmental stages. In parallel, we purified many of the tagged proteins from embryos using the affinity tags and identified co-purifying proteins by mass spectrometry. The fly stocks are publicly available through the Kyoto Drosophila Genetics Resource Center. All our data are available via an open access database (Flannotator), which provides comprehensive information on the expression patterns, subcellular localisations and in vivo interaction partners of the trapped proteins. Our resource substantially increases the number of available protein traps in Drosophila and identifies new markers for cellular organelles and structures.This work was supported by a project grant from the Wellcome Trust [076739], by a Wellcome Trust Principal Research Fellowship to D.StJ. [049818 and 080007], and by core support from the Wellcome Trust [092096] and Cancer Research UK [A14492].This is the final version of the article. It was first available from The Company of Biologists via http://dx.doi.org/10.1242/dev.11105

A transcriptional response to replication stress selectively expands a subset of Brca2 -mutant mammary epithelial cells

Acknowledgements: We thank Dr. Ben Hall (UCL) and Dr. Harveer Dev (Department of Oncology, Cambridge) for critical reading of our manuscript. We thank the CIMR flow cytometry facility for assistance with cell sorting and the CRUK Cambridge Institute genomics core facility, in particular Katarzyna Kania with preparing the samples for single-cell RNA sequencing, and the DFCI Mass Cytometry Core, led by Nicole Paul and Eric Haas. This work was funded by Gray Foundation Team Science Award to J.S.B. and A.R.V., Medical Research Council (MRC) Programme grants MC_UU_12022/1 and MC_UU_12022/8 to A.R.V., and by the Krishnan-Ang Fellowship to M.S.Funder: Krishnan-Ang FellowshipGermline BRCA2 mutation carriers frequently develop luminal-like breast cancers, but it remains unclear how BRCA2 mutations affect mammary epithelial subpopulations. Here, we report that monoallelic Brca2mut/WT mammary organoids subjected to replication stress activate a transcriptional response that selectively expands Brca2mut/WT luminal cells lacking hormone receptor expression (HR-). While CyTOF analyses reveal comparable epithelial compositions among wildtype and Brca2mut/WT mammary glands, Brca2mut/WT HR- luminal cells exhibit greater organoid formation and preferentially survive and expand under replication stress. ScRNA-seq analysis corroborates the expansion of HR- luminal cells which express elevated transcript levels of Tetraspanin-8 (Tspan8) and Thrsp, plus pathways implicated in replication stress survival including Type I interferon responses. Notably, CRISPR/Cas9-mediated deletion of Tspan8 or Thrsp prevents Brca2mut/WT HR- luminal cell expansion. Our findings indicate that Brca2mut/WT cells activate a transcriptional response after replication stress that preferentially favours outgrowth of HR- luminal cells through the expression of interferon-responsive and mammary alveolar genes