Cell surface signalling laboratory

The Cell surface signalling laboratory is interested in the molecular basis of cellular recognition. Using our technologies, we have shown that it is possible to identify extracellular protein interactions that are essential for cellular processes that are critical for disease pathogenesis. We are currently working on recognition events that are important in malaria, heart disease and fertilization.

The team, headed by Gavin Wright, has developed a screening assay called AVEXIS (AVidity-based EXtracellular Interaction Screen) that overcomes many of the technical problems associated with identifying interactions between receptor proteins. Using this method, the team takes a unique large-scale approach to systematically identify networks of extracellular protein interactions between cells.

The basic rationale is to compile recombinant protein libraries containing the full repertoire of receptor proteins displayed on the surface of known interacting cell types. These receptor proteins are then screened against each other to identify novel interactions. The cellular interactions that we select for study are important for fundamental biological processes or the pathogenesis of disease. Once we have identified the receptor-ligand interactions involved, we aim to determine their role using functional assays.

Taking this approach, we have identified critical receptor interactions that can then be targeted to develop novel therapeutics. Importantly, extracellular protein interactions are amenable to this approach because they are accessible to systemically delivered drugs such as therapeutic antibodies. Currently, the laboratory is focusing on three main cellular interactions:

  • P. falciparum merozoite:Red blood cell - A host-pathogen interaction required for the pathogenesis of malaria.
  • Platelet:Platelet - The cellular interaction responsible for heart disease and stroke, two of the leading causes of human mortality.
  • Sperm:Egg - The cellular interaction responsible for new life. Understanding this could lead to the development of novel contraceptives and fertility treatments.

[Anne Weston, LRI, CRUK, Wellcome Images]

Technology

Cells can interact and communicate with each other through specific protein interactions made by cell surface receptor proteins embedded within the membrane that surrounds each cell. Many of the instructive signals received by cells originate from their immediate environment, the cell surface. More specifically, signals are sent and received by membrane proteins: special proteins that bridge the plasma membrane and are able to convey extracellular binding events within the cell. It is these proteins, and especially the extracellular interactions that they make with other proteins, that are the main interest of the laboratory. We aim to discover entirely new signalling pathways by identifying novel cell surface receptor-ligand pairs. Despite the known importance of these interactions in biology, relatively few cell surface proteins have known binding partners because they are biochemically difficult to manipulate.

AVEXIS

How AVEXIS resolves extracellular protein interaction networks.

How AVEXIS resolves extracellular protein interaction networks. [Genome Research Limited]

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Extracellular protein interactions made by membrane-embedded receptors are challenging to study because the proteins are biochemically difficult to work with: they are relatively rare (103/105 copies per cell), hard to solubilise in their native conformation and often contain structurally-critical posttranslational modifications such as disulfide bonds and glycosylation. In addition, the interactions mediated by these receptor proteins are often extremely transient, having half-lives of less than a second.

To circumvent the biochemical difficulties of working with membrane-embedded receptors, we have developed an assay called AVEXIS (for AVidity-based EXtracellular Interaction Screen) which can detect low affinity extracellular protein interactions in a convenient and scalable format. The approach works by compiling protein libraries constituting the whole ectodomain of a cell's receptor repertoire expressed as soluble recombinant proteins in mammalian cells. This enables us to preserve the extracellular binding activity of the receptor but in a format that allows us to manipulate the proteins easily.

The ectodomains are expressed as a bait and/or prey: the baits are biotinylated and captured on streptavidin-coated solid phases (such as a glass slide or microtitre plate) and the preys are pentamerised and enzyme-tagged. By pentamerising the prey proteins, we increase the local concentration of the ectodomain which increases the overall binding strength (avidity) of the interaction enabling us to then detect even very transient (t1/2 <= 0.1sec) interactions with a low false positive rate. The assay is highly adaptable and can be used on microtitre plates and also protein microarrays. The approach depends upon the production of protein libraries that represent the repertoire of receptors present on the interacting cell types.

Research

We focus on three key areas of cell surface signalling:

A merozoite invading an erythrocyte.

A merozoite invading an erythrocyte. [Miller LH et al., 1979. Originally published in Journal of Experimental Medicine. doi:10.1084/jem.149.1.172]

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Malaria - P. falciparum merozoite:red blood cell interactions

Malaria kills around 1 million people every year (mostly children in sub-Saharan Africa) and remains a major global health burden.

It is an infectious disease caused by parasites belonging to the Plasmodium genus and infections by P. falciparum are responsible for the vast majority of mortality. All the clinical symptoms of malaria result from the blood stages of infection which are initiated when the merozoite - the invasive form of the parasite - recognizes and invades a host red blood cell.

We have compiled protein libraries that represent the cell surface and secreted protein repertoire of the P. falciparum merozoite and human erythrocyte and used them in our screening approach to identify novel receptor-ligand interactions involved in invasion. We have tested these interactions in a close collaboration with Julian Rayner's group at the Sanger Institute. Together, we have identified novel receptor-ligand interactions that are important for invasion and are now using these findings to develop new anti-malarial drugs.

Coloured scanning electron micrograph (SEM) of a clump of activated platelets from a blood clot.

Coloured scanning electron micrograph (SEM) of a clump of activated platelets from a blood clot. [Steve Gschmeissner/Science Photo Library]

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Cardiovascular disease - Platelet:platelet interactions

Heart disease and stroke (together known as cardiovascular diseases) are the number one cause of human mortality, responsible for approximately 30 per cent of all deaths globally.

These diseases are caused by the inappropriate aggregation of platelets to form a thrombus, occluding the blood supply to vital organs such as the heart and brain. Platelets aggregate through the adhesive interactions of the receptor proteins displayed on their cell surface but no systematic survey of these interactions has been performed.

By compiling a recombinant protein library containing whole ectodomain fragments of approximately 150 human platelet receptor proteins, we are aiming to use our AVEXIS and "protein chip" technology to identify novel receptor pairs that are important in platelet aggregation. By taking this systematic large-scale approach, we hope to gain an integrated, global view of platelet adhesion and provide new avenues for therapeutic intervention.

Fertilization occurs when a sperm cell recognises and fuses with an egg.

Fertilization occurs when a sperm cell recognises and fuses with an egg. [istockphoto.com]

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Fertilisation - Sperm:oocyte interactions

Most life begins when haploid gametes join to form a diploid zygote; in humans, this happens when a sperm recognizes and fuses with an egg. Fertilization is arguably the most important cell recognition event in biology, yet surprisingly the molecular interactions that underpin the recognition and fusion of gametes are not characterised.

We have compiled both sperm and egg receptor protein libraries, and by recombinantly expressing the extracellular domains of these proteins it is possible to screen for low affinity interactions between cell surface receptors using AVEXIS, and thus discover unknown protein-protein interactions. Concomitant in vitro fertilization assays are used to determine the functional relevance of these newly discovered interactions. By gaining a better insight to the molecular mechanisms of fertilization, we hope to identify novel contraceptive targets and enhance our understanding of infertility.

Resources

Database

ARNIE - AVEXIS Receptor Network with Integrated Expression

Selected Publications

  • A benchmarked protein microarray-based platform for the identification of novel low-affinity extracellular protein interactions.

    Sun Y, Gallagher-Jones M, Barker C and Wright GJ

    Analytical biochemistry 2012;424;1;45-53

  • Basigin is a receptor essential for erythrocyte invasion by Plasmodium falciparum.

    Crosnier C, Bustamante LY, Bartholdson SJ, Bei AK, Theron M, Uchikawa M, Mboup S, Ndir O, Kwiatkowski DP, Duraisingh MT, Rayner JC and Wright GJ

    Nature 2011;480;7378;534-7

  • Jamb and jamc are essential for vertebrate myocyte fusion.

    Powell GT and Wright GJ

    PLoS biology 2011;9;12;e1001216

  • DeltaC and DeltaD interact as Notch ligands in the zebrafish segmentation clock.

    Wright GJ, Giudicelli F, Soza-Ried C, Hanisch A, Ariza-McNaughton L and Lewis J

    Development (Cambridge, England) 2011;138;14;2947-56

  • The blood-stage malaria antigen PfRH5 is susceptible to vaccine-inducible cross-strain neutralizing antibody.

    Douglas AD, Williams AR, Illingworth JJ, Kamuyu G, Biswas S, Goodman AL, Wyllie DH, Crosnier C, Miura K, Wright GJ, Long CA, Osier FH, Marsh K, Turner AV, Hill AV and Draper SJ

    Nature communications 2011;2;601

  • Construction of a large extracellular protein interaction network and its resolution by spatiotemporal expression profiling.

    Martin S, Söllner C, Charoensawan V, Adryan B, Thisse B, Thisse C, Teichmann S and Wright GJ

    Molecular & cellular proteomics : MCP 2010;9;12;2654-65

  • A rapid and scalable method for selecting recombinant mouse monoclonal antibodies.

    Crosnier C, Staudt N and Wright GJ

    BMC biology 2010;8;76

  • Signal initiation in biological systems: the properties and detection of transient extracellular protein interactions.

    Wright GJ

    Molecular bioSystems 2009;5;12;1405-12

  • A cell surface interaction network of neural leucine-rich repeat receptors.

    Söllner C and Wright GJ

    Genome biology 2009;10;9;R99

  • Large-scale screening for novel low-affinity extracellular protein interactions.

    Bushell KM, Söllner C, Schuster-Boeckler B, Bateman A and Wright GJ

    Genome research 2008;18;4;622-30

  • Lymphoid/neuronal cell surface OX2 glycoprotein recognizes a novel receptor on macrophages implicated in the control of their function.

    Wright GJ, Puklavec MJ, Willis AC, Hoek RM, Sedgwick JD, Brown MH and Barclay AN

    Immunity 2000;13;2;233-42

Team

Team members

Josefin Bartholdson Scott
jb28@sanger.ac.ukPostdoctoral Fellow
Enrica Bianchi
Postdoctoral Fellow
Nicole Muller-Sienerth
nms@sanger.ac.ukLaboratory Manager
Abi Perrin
PhD Student
Gareth Powell
gp3@sanger.ac.ukunknown
Nicole Staudt
ns8@sanger.ac.ukProject Leader
Zenon Zenonos
Postdoctoral Fellow

Josefin Bartholdson Scott

jb28@sanger.ac.uk Postdoctoral Fellow

I graduated from the University of Edinburgh in 2004 with a BSc Honours degree in Microbiology and Infection. I continued my studies in Edinburgh with a PhD in Biological Chemistry working on virulence factors of the multi-resistant cystic fibrosis bacteria Burkholderia cenocepacia. I joined the Sanger Institute in 2009.

Research

I am currently investigating novel interactions between Plasmodium proteins involved in malaria infection and receptors on the erythrocyte surface.

References

  • Basigin is a receptor essential for erythrocyte invasion by Plasmodium falciparum.

    Crosnier C, Bustamante LY, Bartholdson SJ, Bei AK, Theron M, Uchikawa M, Mboup S, Ndir O, Kwiatkowski DP, Duraisingh MT, Rayner JC and Wright GJ

    Cell Surface Signalling Laboratory, Wellcome Trust Sanger Institute, Hinxton, Cambridge CB10 1HH, UK.

    Erythrocyte invasion by Plasmodium falciparum is central to the pathogenesis of malaria. Invasion requires a series of extracellular recognition events between erythrocyte receptors and ligands on the merozoite, the invasive form of the parasite. None of the few known receptor-ligand interactions involved are required in all parasite strains, indicating that the parasite is able to access multiple redundant invasion pathways. Here, we show that we have identified a receptor-ligand pair that is essential for erythrocyte invasion in all tested P. falciparum strains. By systematically screening a library of erythrocyte proteins, we have found that the Ok blood group antigen, basigin, is a receptor for PfRh5, a parasite ligand that is essential for blood stage growth. Erythrocyte invasion was potently inhibited by soluble basigin or by basigin knockdown, and invasion could be completely blocked using low concentrations of anti-basigin antibodies; importantly, these effects were observed across all laboratory-adapted and field strains tested. Furthermore, Ok(a-) erythrocytes, which express a basigin variant that has a weaker binding affinity for PfRh5, had reduced invasion efficiencies. Our discovery of a cross-strain dependency on a single extracellular receptor-ligand pair for erythrocyte invasion by P. falciparum provides a focus for new anti-malarial therapies.

    Funded by: Medical Research Council: G19/9; NCEZID CDC HHS: R36 CK000119-01; NIAID NIH HHS: 2T32 AI007535-12, R01 AI057919, R01 AI057919-05, R01AI057919; Wellcome Trust: 077108, 089084, 090532

    Nature 2011;480;7378;534-7

  • Contributions of two UDP-glucose dehydrogenases to viability and polymyxin B resistance of Burkholderia cenocepacia.

    Loutet SA, Bartholdson SJ, Govan JR, Campopiano DJ and Valvano MA

    Department of Microbiology and Immunology, Infectious Diseases Research Group, Siebens-Drake Research Institute, University of Western Ontario, London, Ontario N6A 5C1, Canada.

    Burkholderia cenocepacia is highly resistant to antimicrobial peptides and we hypothesized that the conversion of UDP-glucose to UDP-glucuronic acid, a reaction catalysed by the enzyme UDP-glucose dehydrogenase (Ugd) would be important for this resistance. The genome of B. cenocepacia contains three predicted ugd genes: ugd(BCAL2946), ugd(BCAM0855) and ugd(BCAM2034), all of which were individually inactivated. Only inactivation of ugd(BCAL2946) resulted in increased sensitivity to polymyxin B and this sensitivity could be overcome when either ugd(BCAL2946) or ugd(BCAM0855) but not ugd(BCAM2034) was expressed from plasmids. The growth of a conditional ugd(BCAL2946) mutant, created in the Deltaugd(BCAM0855) background, was significantly impaired under non-permissive conditions. Growth could be rescued by either ugd(BCAL2946) or ugd(BCAM0855) expressed in trans, but not by ugd(BCAM2034). Biochemical analysis of the purified, recombinant forms of Ugd(BCAL2946) and Ugd(BCAM0855) revealed that they are soluble homodimers with similar in vitro Ugd activity and comparable kinetic constants for their substrates UDP-glucose and NAD(+). Purified Ugd(BCAM2034) showed no in vitro Ugd activity. Real-time PCR analysis showed that the expression of ugd(BCAL2946) was 5.4- and 135-fold greater than that of ugd(BCAM0855) and ugd(BCAM2034), respectively. Together, these data indicate that the combined activity of Ugd(BCAL2946) and Ugd(BCAM0855) is essential for the survival of B. cenocepacia but only the most highly expressed ugd gene, ugd(BCAL2946), is required for polymyxin B resistance.

    Microbiology (Reading, England) 2009;155;Pt 6;2029-39

  • Effective binding and sensing of lipopolysaccharide: combining complementary pattern recognition receptors.

    Ganesh V, Bodewits K, Bartholdson SJ, Natale D, Campopiano DJ and Mareque-Rivas JC

    School of Chemistry, University of Edinburgh, Edinburgh EH9 3JJ, UK.

    Angewandte Chemie (International ed. in English) 2009;48;2;356-60

  • Plant host and sugar alcohol induced exopolysaccharide biosynthesis in the Burkholderia cepacia complex.

    Bartholdson SJ, Brown AR, Mewburn BR, Clarke DJ, Fry SC, Campopiano DJ and Govan JR

    School of Chemistry, University of Edinburgh, Edinburgh, UK.

    The species that presently constitute the Burkholderia cepacia complex (Bcc) have multiple roles; they include soil and water saprophytes, bioremediators, and plant, animal and human pathogens. Since the first description of pathogenicity in the Bcc was based on sour skin rot of onion bulbs, this study returned to this plant host to investigate the onion-associated phenotype of the Bcc. Many Bcc isolates, which were previously considered to be non-mucoid, produced copious amounts of exopolysaccharide (EPS) when onion tissue was provided as the sole nutrient. EPS production was not species-specific, was observed in isolates from both clinical and environmental sources, and did not correlate with the ability to cause maceration of onion tissue. Chemical analysis suggested that the onion components responsible for EPS induction were primarily the carbohydrates sucrose, fructose and fructans. Additional sugars were investigated, and all alcohol sugars tested were able to induce EPS production, in particular mannitol and glucitol. To investigate the molecular basis for EPS biosynthesis, we focused on the highly conserved bce gene cluster thought to be involved in cepacian biosynthesis. We demonstrated induction of the bce gene cluster by mannitol, and found a clear correlation between the inability of representatives of the Burkholderia cenocepacia ET12 lineage to produce EPS and the presence of an 11 bp deletion within the bceB gene, which encodes a glycosyltransferase. Insertional inactivation of bceB in Burkholderia ambifaria AMMD results in loss of EPS production on sugar alcohol media. These novel and surprising insights into EPS biosynthesis highlight the metabolic potential of the Bcc and show that a potential virulence factor may not be detected by routine laboratory culture. Our results also highlight a potential hazard in the use of inhaled mannitol as an osmolyte to improve mucociliary clearance in individuals with cystic fibrosis.

    Funded by: Biotechnology and Biological Sciences Research Council

    Microbiology (Reading, England) 2008;154;Pt 8;2513-21

  • Temperate bacteriophages DK4 and BcepMu from Burkholderia cenocepacia J2315 are identical.

    Langley RJ, Kenna D, Bartholdson J, Campopiano DJ and Govan JR

    Cystic Fibrosis Laboratory, Medical Microbiology, University of Edinburgh, Medical School, Teviot Place, EH8 9AG, United Kingdom.

    FEMS immunology and medical microbiology 2005;45;2;349-50

Enrica Bianchi

- Postdoctoral Fellow

I graduated from the University of Bologna (Italy) with a degree in Biological Science and earned my Ph.D. in Reproduction and Development at the University of Rome “Tor Vergata” in 2008. During the following two years as a postdoc I investigated the reproductive defects in mice that were knocked-out for a gene encoding for an RNA-binding protein. In 2011 I moved to the Wellcome Trust Sanger Institute where I joined the Wright's group to study the sperm-egg interaction.

Research

Surprisingly the detailed mechanism of fertilization is far to be elucidated. Which and how many proteins are involved in the recognition, binding and fusion of the sperm with the egg is largely a mystery. Applying the large-scale systematic approaches to identify novel receptor-ligand pairs that have been developed in Wright's lab, this project aims to identify the proteins involved in the sperm-egg interaction. Interestingly this research could help filling the gap in this field and have relevant applications considering that no novel contraceptive has been developed since 1970.

References

  • FLASH is essential during early embryogenesis and cooperates with p73 to regulate histone gene transcription.

    De Cola A, Bongiorno-Borbone L, Bianchi E, Barcaroli D, Carletti E, Knight RA, Di Ilio C, Melino G, Sette C and De Laurenzi V

    IDI-IRCCS Biochemistry Laboratory, c/o Department of Experimental Medicine, University of Rome Tor Vergata, Roma, Italy.

    Replication-dependent histone gene expression is a fundamental process occurring in S-phase under the control of the cyclin-E/CDK2 complex. This process is regulated by a number of proteins, including Flice-Associated Huge Protein (FLASH) (CASP8AP2), concentrated in specific nuclear organelles known as HLBs. FLASH regulates both histone gene transcription and mRNA maturation, and its downregulation in vitro results in the depletion of the histone pull and cell-cycle arrest in S-phase. Here we show that the transcription factor p73 binds to FLASH and is part of the complex that regulates histone gene transcription. Moreover, we created a novel gene trap to disrupt FLASH in mice, and we show that homozygous deletion of FLASH results in early embryonic lethality, owing to arrest of FLASH(-/-) embryos at the morula stage. These results indicate that FLASH is an essential, non-redundant regulator of histone transcription and cell cycle during embryogenesis.

    Funded by: Medical Research Council: MC_U132670600; Telethon: GGP09133

    Oncogene 2012;31;5;573-82

  • Ablation of the Sam68 gene impairs female fertility and gonadotropin-dependent follicle development.

    Bianchi E, Barbagallo F, Valeri C, Geremia R, Salustri A, De Felici M and Sette C

    Department of Public Health and Cell Biology, University of Rome Tor Vergata, 00133 Rome, Italy.

    Sam68 is a multifunctional RNA-binding protein highly expressed in the gonads, whose ablation causes male infertility. Herein, we have investigated Sam68 expression in the adult ovary and its function in female fertility. Immunohistochemistry showed that Sam68 was localized in the nucleus of oocytes and follicular cells at all stages of folliculogenesis. Sam68(-/-) females were severely subfertile, and they showed a delay in the age of first pregnancy, increased breeding time for successful pregnancy and yielded smaller litters. Morphological analyses indicated a significant reduction in the number of secondary and pre-antral follicles in the ovary. These defects were associated with alteration of oestrous cycles and a reduced number of ovulated oocytes, which were only partially restored by the administration of exogenous gonadotropins. Crosslinking/immunoprecipitation experiments showed that Sam68 directly binds the mRNAs for the follicle-stimulating hormone (FSH) and the luteinizing hormone receptors (Fshr and Lhcgr), which were downregulated in ovaries of adult knockout females. Stimulation of immature females with FSH-like pregnant mare serum gonadotropin (PMSG), or of follicular cells with the FSH second messenger analogue 8Br-cAMP, caused the upregulation of Sam68. The increase in Sam68 levels paralleled that of the Fshr and Lhcgr mRNAs in the pre-ovulatory follicle and was required to allow accumulation of these transcripts in follicular cells. These studies identify a new crucial function for Sam68 in the regulation of female fertility and indicate that this protein is required to insure proper expression of the gonadotropin receptor transcripts in pre-ovulatory follicles in adult ovary.

    Funded by: Telethon: GGP09154

    Human molecular genetics 2010;19;24;4886-94

  • Expression of stemness markers in mouse parthenogenetic-diploid blastocysts is influenced by slight variation of activation protocol adopted.

    Bianchi E, Geremia R and Sette C

    Department of Public Health and Cell Biology, Section of Anatomy, University of Rome "Tor Vergata", Via Montpellier 1, 00133 Rome, Italy.

    The importance of obtaining stem cells through alternative methods has increased progressively in the recent years due to the potential role that embryonic stem (ES) cells play in the field of regenerative medicine. In this regard, generation of parthenogenetic blastocysts allows the production of ethic-free ES cells without the need to manipulate normal embryos. Our work was aimed at clarifying whether variations in the method adopted to generate diploid parthenogenetic blastocysts could determine differences in the quality of blastocysts produced. In vitro development of mouse oocytes activated with three protocols, using Sr2+ and cytochalasin for different time, was compared with that of in vivo fertilized embryos. We have evaluated the efficiency of blastocyst formation and analysed the expression pattern of the stemness markers OCT4, CDX2, and NANOG. Our results indicate that the yield of diploid parthenogenotes and the segregation of the stemness marker OCT4 in the developing blastocyst are influenced by the parthenogenetic protocol adopted. Particularly, even if all methods tested allowed the production of blastocysts in vitro, the correct segregation of OCT4 occurred only in blastocysts developed from oocytes concomitantly treated for 4 h with Sr2+ and cytochalasin D. Our results indicate that the protocol employed to develop parthenogenetic blastocysts in vitro affects the quality of cells in the inner cell mass.

    Funded by: Telethon: GGP09154

    In vitro cellular & developmental biology. Animal 2010;46;7;619-23

  • Sam68 regulates translation of target mRNAs in male germ cells, necessary for mouse spermatogenesis.

    Paronetto MP, Messina V, Bianchi E, Barchi M, Vogel G, Moretti C, Palombi F, Stefanini M, Geremia R, Richard S and Sette C

    Department of Public Health and Cell Biology, Section of Anatomy, University of Rome Tor Vergata, 00133 Rome, Italy.

    Sam68 is a KH-type RNA-binding protein involved in several steps of RNA metabolism with potential implications in cell differentiation and cancer. However, its physiological roles are still poorly understood. Herein, we show that Sam68(-/-) male mice are infertile and display several defects in spermatogenesis, demonstrating an essential role for Sam68 in male fertility. Sam68(-/-) mice produce few spermatozoa, which display dramatic motility defects and are unable to fertilize eggs. Expression of a subset of messenger mRNAs (mRNAs) is affected in the testis of knockout mice. Interestingly, Sam68 is associated with polyadenylated mRNAs in the cytoplasm during the meiotic divisions and in round spermatids, when it interacts with the translational machinery. We show that Sam68 is required for polysomal recruitment of specific mRNAs and for accumulation of the corresponding proteins in germ cells and in a heterologous system. These observations demonstrate a novel role for Sam68 in mRNA translation and highlight its essential requirement for the development of a functional male gamete.

    Funded by: Telethon: GGP04118

    The Journal of cell biology 2009;185;2;235-49

  • Dynamic expression of the RNA-binding protein Sam68 during mouse pre-implantation development.

    Paronetto MP, Bianchi E, Geremia R and Sette C

    Department of Public Health and Cell Biology, Section of Anatomy, University of Rome Tor Vergata, Via Montpellier 1, 00133 Rome, Italy.

    The STAR protein Sam68 (KHDRBS1) is involved in several aspects of post-transcriptional mRNA metabolism. Herein, we have investigated the expression and subcellular localization of Sam68 during early mouse embryogenesis. We found that mouse oocytes express high levels of Sam68 mRNA, low levels of the transcript for Khdrbs2 (current symbol for Slm-1) and no Khdrbs3 (current symbol for Slm-2), two highly homologous STAR genes. Sam68 protein is expressed throughout oocyte meiotic maturation and early embryogenesis. It is released in the cytoplasm upon meiotic resumption and it slowly accumulates in the nucleus after fertilization. Unlike what was observed for other RNA-binding proteins, nuclear accumulation of Sam68 was independent of de novo mRNA transcription. However, we found that inhibition of mRNA translation by either cycloheximide or puromycin in one-cell embryos caused the accumulation of Sam68 in cytoplasmic granules. Analysis of these granules by deconvolution microscopy demonstrated that they are sites of accumulation for proteins involved in the initiation of mRNA translation, such as eIF4A1, eIF4E and eIF4G. These granules contained RNA and were dissolved by treatment with RNase A. Other proteins expressed by the zygote, like the splicing factor SC35 or the cytoplasmic kinase ERK2, did not accumulate in such structures after treatment with inhibitors of mRNA translation, indicating that the localization of Sam68 and of the translation initiation factors in these granules is a specific event. These results indicate that Sam68 is involved in translational regulation of maternal mRNAs in the zygote and in the early signaling events triggered by fertilization.

    Funded by: Telethon: GGP04118

    Gene expression patterns : GEP 2008;8;5;311-22

Nicole Muller-Sienerth

nms@sanger.ac.uk Laboratory Manager

I studied Biology at the University of Hohenheim (Germany) and completed my Diploma (MSc) in Microbiology in 2004. I finished my PhD in 2009 and continued working as a PostDoc with Prof. Harald Wajant at the University Hospital of Wuerzburg (Germany) in the research field of Tumor Necrosis Factor (TNF) ligands and receptors. I joined Gavin Wright's Cell surface signalling laboratory as an Advanced Research Assistant in 2011.

Research

The target of my current project is to clone recombinant monoclonal antibodies against extracellular domains of 200 different zebrafish cell surface and secreted proteins. We immunise mice and thereof generate hybridomas producing antibodies against our antigens. Afterwards we use a specific cloning strategy to develop recombinant versions of these specific antibodies. They will be further tested by performing immunocytochemistry to stain wholemount embryos and cryosections.

Or aim is to distribute this antibody recourse freely and widely to the zebrafish community.

References

  • Genetic engineering of death ligands for improvement of therapeutic activity.

    Gerspach J, Schneider B, Müller N, Otz T, Wajant H and Pfizenmaier K

    Institute of Cell Biology and Immunology, University of Stuttgart, Stuttgart, Germany.

    Advances in experimental medicine and biology 2011;691;507-19

  • SMAC mimetic BV6 induces cell death in monocytes and maturation of monocyte-derived dendritic cells.

    Müller-Sienerth N, Dietz L, Holtz P, Kapp M, Grigoleit GU, Schmuck C, Wajant H and Siegmund D

    Department of Internal Medicine II, University Hospital Würzburg, Würzburg, Germany.

    Background: Compounds mimicking the inhibitory effect of SMAC/DIABLO on X-linked inhibitor of apoptosis (XIAP) have been developed with the aim to achieve sensitization for apoptosis of tumor cells resistant due to deregulated XIAP expression. It turned out that SMAC mimetics also have complex effects on the NFκB system and TNF signaling. In view of the overwhelming importance of the NFκB transcription factors in the immune system, we analyzed here the effects of the SMAC mimetic BV6 on immune cells.

    BV6 induced apoptotic and necrotic cell death in monocytes while T-cells, dendritic cells and macrophages were largely protected against BV6-induced cell death. In immature dendritic cells BV6 treatment resulted in moderate activation of the classical NFκB pathway, but it also diminished the stronger NFκB-inducing effect of TNF and CD40L. Despite its inhibitory effect on TNF- and CD40L signaling, BV6 was able to trigger maturation of immature DCs as indicated by upregulation of CD83, CD86 and IL12.

    Significance: The demonstrated effects of SMAC mimetics on immune cells may complicate the development of tumor therapeutic concepts based on these compounds but also arise the possibility to exploit them for the development of immune stimulatory therapies.

    PloS one 2011;6;6;e21556

  • Superior serum half life of albumin tagged TNF ligands.

    Müller N, Schneider B, Pfizenmaier K and Wajant H

    Division of Molecular Internal Medicine, Department of Internal Medicine II, University Hospital Würzburg, Röntgenring 11, 97070 Würzburg, Germany.

    Due to their immune stimulating and apoptosis inducing properties, ligands of the TNF family attract increasing interest as therapeutic proteins. A general limitation of in vivo applications of recombinant soluble TNF ligands is their notoriously rapid clearance from circulation. To improve the serum half life of the TNF family members TNF, TWEAK and TRAIL, we genetically fused soluble variants of these molecules to human serum albumin (HSA). The serum albumin-TNF ligand fusion proteins were found to be of similar bioactivity as the corresponding HSA-less counterparts. Upon intravenous injection (i.v.), serum half life of HSA-TNF ligand fusion proteins, as determined by ELISA, was around 15 h as compared to approximately 1h for all of the recombinant control TNF ligands without HSA domain. Moreover, serum samples collected 6 or 24h after i.v. injection still contained high TNF ligand bioactivity, demonstrating that there is only limited degradation/inactivation of circulating HSA-TNF ligand fusion proteins in vivo. In a xenotransplantation model, significantly less of the HSA-TRAIL fusion protein compared to the respective control TRAIL protein was required to achieve inhibition of tumor growth indicating that the increased half life of HSA-TNF ligand fusion proteins translates into better therapeutic action in vivo. In conclusion, our data suggest that genetic fusion to serum albumin is a powerful and generally applicable mean to improve bioavailability and in vivo activity of TNF ligands.

    Biochemical and biophysical research communications 2010;396;4;793-9

  • Membrane tumor necrosis factor (TNF) induces p100 processing via TNF receptor-2 (TNFR2).

    Rauert H, Wicovsky A, Müller N, Siegmund D, Spindler V, Waschke J, Kneitz C and Wajant H

    Division of Molecular Internal Medicine, Department of Internal Medicine II, University Hospital Würzburg, Röntgenring 11, 97070 Würzburg, Germany.

    Tumor necrosis factor (TNF) elicits its biological activities by stimulation of two receptors, TNFR1 and TNFR2, both belonging to the TNF receptor superfamily. Whereas TNFR1-mediated signal transduction has been intensively studied and is understood in detail, especially with respect to activation of the classical NFkappaB pathway, cell death induction, and MAP kinase signaling, TNFR2-associated signal transduction is poorly defined. Here, we demonstrate in various tumor cell lines and primary T-cells that TNFR2, but not TNFR1, induces activation of the alternative NFkappaB pathway. In accord with earlier findings demonstrating that only membrane TNF, but not soluble TNF, properly activates TNFR2, we further show by use of TNFR1- and TNFR2-specific mutants of soluble TNF and membrane TNF that soluble ligand trimers fail to activate the alternative NFkappaB pathway. In accord with the known inhibitory role of TRAF2 in the alternative NFkappaB pathway, TNFR2-, but not TNFR1-specific TNF induced depletion of cytosolic TRAF2. Thus, we identified activation of the alternative NFkappaB pathway as a TNF signaling effect that can be specifically assigned to TNFR2 and membrane TNF.

    The Journal of biological chemistry 2010;285;10;7394-404

  • Trimer stabilization, oligomerization, and antibody-mediated cell surface immobilization improve the activity of soluble trimers of CD27L, CD40L, 41BBL, and glucocorticoid-induced TNF receptor ligand.

    Wyzgol A, Müller N, Fick A, Munkel S, Grigoleit GU, Pfizenmaier K and Wajant H

    Division of Molecular Internal Medicine, Department of Internal Medicine II, University Hospital Wuerzburg, Wuerzburg, Germany.

    For many ligands of the TNF family, trimer stability and oligomerization status are crucial determinants of receptor activation. However, for the immunostimulatory ligands CD27L, CD40L, 41BBL, and glucocorticoid-induced TNF receptor ligand (GITRL) detailed information regarding these requirements is lacking. Here, we comprehensively evaluated the effect of trimer stability and oligomerization on receptor activation by these ligands. Treatment with soluble Flag-tagged CD27L, 41BBL, and GITRL minimally activated receptor signaling, while Flag-CD40L was highly active. Oligomerization with anti-Flag Abs further enhanced the specific activity of Flag-CD40L 10-fold and of Flag-41BBL more than 200-fold, but it failed to activate Flag-CD27L and Flag-GITRL. We next investigated the relevance of trimer stability by introducing the tenascin-C (TNC) trimerization domain, yielding stabilized Flag-TNC-ligand trimers. Oligomerization with anti-Flag Ab potently activated signaling by Flag-TNC-CD27L and Flag-TNC-GITRL and, albeit to a lesser extent, Flag-TNC-CD40L and Flag-TNC-41BBL. Forced hexamerization, by introducing an Ig Fc domain, revealed that hexameric derivatives of Flag-TNC-41BBL, Flag-CD40L, and Flag-TNC-GITRL all activate receptor signaling with high efficiency, whereas hexameric Flag-CD27L variant left inactive. Finally, we attempted to selectively activate receptor signaling on targeted cells, by using Ab fragment (single-chain fragment variable region, scFv)-ligand fusion proteins, an approach previously applied to other TNF ligands. Target cell surface Ag-selective activation was achieved for scFv-41BBL, scFv-CD40L, and scFv-GITRL, although the latter two displayed already significant activity toward Ag-negative cells. In conclusion, our data establish that trimeric CD40L is active, 41BBL requires hexamerization, GITRL requires trimer stabilization, and CD27L requires trimer stabilization and oligomerization. Furthermore, surface immobilization might be exploited to gain locally enhanced ligand activity.

    Journal of immunology (Baltimore, Md. : 1950) 2009;183;3;1851-61

  • Activity of soluble OX40 ligand is enhanced by oligomerization and cell surface immobilization.

    Müller N, Wyzgol A, Münkel S, Pfizenmaier K and Wajant H

    Department of Molecular Internal Medicine, Medical Clinic and Polyclinic II, University of Wuerzburg, Germany.

    OX40 ligand (OX40L) and OX40 are typical members of the tumor necrosis factor ligand family and the tumor necrosis factor receptor superfamily, respectively, and are involved in the costimulation and differentiation of T cells. Like other tumor necrosis factor ligands, OX40L is a type II transmembrane protein. Recombinant soluble human OX40L assembles into trimers and is practically inactive despite binding to OX40. However, oligomerization of soluble OX40L trimers by cross-linking with antibodies or by expression as a hexameric fusion protein strongly increased the activity of the ligand. Moreover, a fusion protein of OX40L with a single chain fragment recognizing the tumor stroma antigen fibroblast activation protein showed a cell surface antigen-dependent increase in the activity of the ligand domain of the molecule and thus mimicked the activity of membrane OX40L upon antigen binding. Trimeric single chain OX40L fusion proteins therefore represent a novel type of OX40L-derived immunostimulatory molecule with potentially reduced systemic side effects.

    The FEBS journal 2008;275;9;2296-304

  • Superior activity of fusion protein scFvRit:sFasL over cotreatment with rituximab and Fas agonists.

    Bremer E, ten Cate B, Samplonius DF, Mueller N, Wajant H, Stel AJ, Chamuleau M, van de Loosdrecht AA, Stieglmaier J, Fey GH and Helfrich W

    Groningen University Institute for Drug Exploration, Department of Pathology and Laboratory Medicine, University Medical Center Groningen, Groningen, the Netherlands.

    The clinical efficacy of the CD20-specific chimeric monoclonal antibody rituximab is significantly hampered by intrinsic or acquired resistance to therapy. Rituximab activates antibody-dependent cellular cytotoxicity/complement-dependent cytotoxicity-dependent lysis but also induces apoptosis by cross-linking of its target antigen CD20. Recent reports indicate that this apoptotic activity of rituximab can be synergized by cotreatment with Fas agonists. Here, we report on a strategy designed to exploit and optimize the synergy between rituximab and Fas signaling by genetically fusing a rituximab-derived antibody fragment to soluble Fas ligand (sFasL). The resultant fusion protein, designated scFvRit:sFasL, potently induced CD20-restricted apoptosis in a panel of malignant B-cell lines (10 of 11) and primary patient-derived malignant B cells (two of two non-Hodgkin lymphoma and five of six B cell chronic lymphocytic leukemia). ScFvRit:sFasL efficiently activated CD20 and Fas apoptotic signaling, resulting in a far superior proapoptotic activity compared with cotreatment with rituximab and Fas agonists. ScFvRit:sFasL lacked activity toward normal human B cells and also lacked systemic toxicity in nude mice with no elevation of aspartate aminotransferase and alanine aminotransferase levels or liver caspase-3 activity. In conclusion, scFvRit:sFasL efficiently activates CD20 and Fas-apoptotic signaling and may be useful for the elimination of malignant B cells.

    Cancer research 2008;68;2;597-604

  • Sustained JNK activation in response to tumor necrosis factor is mediated by caspases in a cell type-specific manner.

    Wicovsky A, Müller N, Daryab N, Marienfeld R, Kneitz C, Kavuri S, Leverkus M, Baumann B and Wajant H

    Department of Molecular Internal Medicine, Medical Clinic and Polyclinic II, University of Würzburg, Röntgenring 11, 97070 Würzburg, Germany.

    In most cell types, tumor necrosis factor (TNF) induces a transient activation of the JNK pathway. However, in NFkappaB-inhibited cells, TNF stimulates also a second sustained phase of JNK activation, which has been implicated in cell death induction. In the present study, we have analyzed the relationship of cell death induction, caspase activity, JNK, and NFkappaB stimulation in the context of TNF signaling in four different cellular systems. In all cases, NFkappaB inhibition enhanced TNF-induced cell death and primed most, but not all, cells for sustained JNK activation. The caspase inhibitor Benzyloxycarbonyl-Val-Ala-Asp(OMe)-fluoromethyl ketone (Z-VAD-fmk) and overexpression of the antiapoptotic proteins FLIP-L and Bcl2 differentially blocked transient and sustained JNK activation in NFkappaB-inhibited KB and HaCaT cells, indicating that the two phases of TNF-induced JNK activation occur at least in these cellular models by different pathways. Although the broad range caspase inhibitor Z-VAD-fmk and the antioxidant butylated hydroxyanisole interfered with TNF-induced cell death to a varying extent in a cell type-specific manner, inhibition of JNK signaling had no or only a very moderate effect. Notably, the JNK inhibitory effect of neither Z-VAD-fmk nor butylated hydroxyanisole was strictly correlated with the capability of these compounds to rescue cells from TNF-induced cell death. Thus, sustained JNK activation by TNF has no obligate role in TNF-induced cell death and is mediated by caspases and reactive oxygen species in a cell type-specific manner.

    The Journal of biological chemistry 2007;282;4;2174-83

Abi Perrin

- PhD Student

I studied Natural Sciences at the University of Cambridge and specialised in Pathology. During this time I worked in several diffrent laboratories in and around Cambridge. One project involved transient transformation of plant leaves, another (here at Sanger) was looking at the evolution of Salmonella bacteria. I also investigated amplification bias in PCR for my third year lab project. During my PhD rotations I have worked in the malaria group and also looked at bacterial genome evolution. Outside of campus I am actively involved in science teaching and outreach (www.chaosscience.org.uk).

Research

I have been screening for protein-protein interactions between human platelets and P. falciparum merozoites. These protein libraries have been used previously in screens to identify erythrocyte invasion pathways in malaria, and to discover novel platelet-platelet interactions. Novel interactions from this screen will be fully characterised, to see if they have a role in the pathogenesis of malaria.

Gareth Powell

gp3@sanger.ac.uk unknown

I am currently a Postdoctoral Fellow in Gavin Wright's laboratory, having completed the Wellcome Trust PhD programme at the Sanger Institute. I graduated from the University of Cambridge as a member of Magdalene College in 2011. My research was centred on zebrafish muscle development.

Previously, I studied for a Bachelors and Masters degree in Biochemistry at Peterhouse, University of Cambridge and undertook research projects in plant biochemistry, nitrogen fixation (at the John Innes in Norwich) and population genetics.

Research

My research is primarily focused on the role of two zebrafish cell surface proteins in muscle development - specifically, their involvement in fusing precursor cells together into a single muscle fibre.

To date, I have shown that the interaction between the two proteins, Jamb and Jamc, is essential for the process of cell fusion. My main interest in the future is discovering what signals are initiated by this binding event and how the process is controlled.

References

  • Jamb and jamc are essential for vertebrate myocyte fusion.

    Powell GT and Wright GJ

    Wellcome Trust Sanger Institute, Hinxton, Cambridge, United Kingdom.

    Cellular fusion is required in the development of several tissues, including skeletal muscle. In vertebrates, this process is poorly understood and lacks an in vivo-validated cell surface heterophilic receptor pair that is necessary for fusion. Identification of essential cell surface interactions between fusing cells is an important step in elucidating the molecular mechanism of cellular fusion. We show here that the zebrafish orthologues of JAM-B and JAM-C receptors are essential for fusion of myocyte precursors to form syncytial muscle fibres. Both jamb and jamc are dynamically co-expressed in developing muscles and encode receptors that physically interact. Heritable mutations in either gene prevent myocyte fusion in vivo, resulting in an overabundance of mononuclear, but otherwise overtly normal, functional fast-twitch muscle fibres. Transplantation experiments show that the Jamb and Jamc receptors must interact between neighbouring cells (in trans) for fusion to occur. We also show that jamc is ectopically expressed in prdm1a mutant slow muscle precursors, which inappropriately fuse with other myocytes, suggesting that control of myocyte fusion through regulation of jamc expression has important implications for the growth and patterning of muscles. Our discovery of a receptor-ligand pair critical for fusion in vivo has important implications for understanding the molecular mechanisms responsible for myocyte fusion and its regulation in vertebrate myogenesis.

    Funded by: Wellcome Trust: 077047/Z/05/Z, 077108/Z/05/Z

    PLoS biology 2011;9;12;e1001216

  • A common MYBPC3 (cardiac myosin binding protein C) variant associated with cardiomyopathies in South Asia.

    Dhandapany PS, Sadayappan S, Xue Y, Powell GT, Rani DS, Nallari P, Rai TS, Khullar M, Soares P, Bahl A, Tharkan JM, Vaideeswar P, Rathinavel A, Narasimhan C, Ayapati DR, Ayub Q, Mehdi SQ, Oppenheimer S, Richards MB, Price AL, Patterson N, Reich D, Singh L, Tyler-Smith C and Thangaraj K

    Department of Biochemistry, Madurai Kamaraj University, Madurai 625 021, India.

    Heart failure is a leading cause of mortality in South Asians. However, its genetic etiology remains largely unknown. Cardiomyopathies due to sarcomeric mutations are a major monogenic cause for heart failure (MIM600958). Here, we describe a deletion of 25 bp in the gene encoding cardiac myosin binding protein C (MYBPC3) that is associated with heritable cardiomyopathies and an increased risk of heart failure in Indian populations (initial study OR = 5.3 (95% CI = 2.3-13), P = 2 x 10(-6); replication study OR = 8.59 (3.19-25.05), P = 3 x 10(-8); combined OR = 6.99 (3.68-13.57), P = 4 x 10(-11)) and that disrupts cardiomyocyte structure in vitro. Its prevalence was found to be high (approximately 4%) in populations of Indian subcontinental ancestry. The finding of a common risk factor implicated in South Asian subjects with cardiomyopathy will help in identifying and counseling individuals predisposed to cardiac diseases in this region.

    Funded by: NHGRI NIH HHS: R01 HG006399; Wellcome Trust: 077009

    Nature genetics 2009;41;2;187-91

  • Look who's talking too: graduates developing skills through communication.

    Tomazou EM and Powell GT

    Wellcome Trust Sanger Institute, Wellcome Trust Genome Campus, Hinxton, Cambridgeshire, CB10 1SA, UK. emt@sanger.ac.uk

    Greater opportunities for young scientists to present their doctoral research to large general audiences will encourage development of transferable skills and involvement in the scientific community. We look at ways students communicate their research and explore the benefits of student-led meetings. The organization of the first Sanger-Cambridge Ph.D. Symposium provides an example of how students can act to establish forums for their work and we call on other young scientists to do the same.

    Nature reviews. Genetics 2007;8;9;724-6

  • The population history of the Xibe in northern China: a comparison of autosomal, mtDNA and Y-chromosomal analyses of migration and gene flow.

    Powell GT, Yang H, Tyler-Smith C and Xue Y

    The Wellcome Trust Sanger Institute, Wellcome Trust Genome Campus, Hinxton, Cambridgeshire CB10 1SA, UK.

    The Xibe population originated in northeastern China, but migrated to northwestern China in 1764-6. We have used a combination of published autosomal and Y-chromosomal data, together with newly derived mtDNA HVSI sequences, to investigate the extent to which genetic data can reveal the geographical origin of this population and the level of gene flow after the migration. We find that mtDNA data are uninformative, but that both autosomal and Y-chromosomal data indicate a northeastern origin, and that the Y data suggest 28% subsequent male-mediated gene flow into the population. We thus conclude that an appropriate combination of genetic data and analytical methods can reveal even recent and local events in the history of a population. In the Chinese samples examined, the combination of a northeastern autosomal background with a northwestern Y chromosome is indicative of a Xibe individual.

    Funded by: Wellcome Trust

    Forensic science international. Genetics 2007;1;2;115-9

Nicole Staudt

ns8@sanger.ac.uk Project Leader

I was awarded with a Diploma in Biology at the University of Marburg (Germany)in 1998. In the same year I started my PhD studies at the MPI for Biophysical Chemistry in Goettingen (Germany). Both my Diploma and my PhD thesis cover certain aspects of skeletal muscle development in the fruitfly Drosophila melanogaster. After moving to London in 2002 I started my postdoc at the MRC Centre for Developmental Neurobiology working on early neural plate development in the zebrafish Danio rerio. Since 2008 I work in the Cell Surface Signalling group at the WTSI as a Project Leader.

Research

Our aim is to generate an antibody resource for zebrafish researchers. We created a library of zebrafish cell surface specific markers in mammalian exression vectors. This enables us to express the proteins in cell cuture in order to immunise mice. After hybridoma fusion and selection of specific antibody producing cells we clone recombinant antibodies in an expression vector. These are tested in vitro and on zebrafish embryos. We started creating our own project specific website in order to make these recombinant antibodies available to the zebrafish community.

References

  • A rapid and scalable method for selecting recombinant mouse monoclonal antibodies.

    Crosnier C, Staudt N and Wright GJ

    Cell Surface Signalling Laboratory, Wellcome Trust Sanger Institute, Cambridge CB10 1HH, UK.

    Background: Monoclonal antibodies with high affinity and selectivity that work on wholemount fixed tissues are valuable reagents to the cell and developmental biologist, and yet isolating them remains a long and unpredictable process. Here we report a rapid and scalable method to select and express recombinant mouse monoclonal antibodies that are essentially equivalent to those secreted by parental IgG-isotype hybridomas.

    Results: Increased throughput was achieved by immunizing mice with pools of antigens and cloning - from small numbers of hybridoma cells - the functionally rearranged light and heavy chains into a single expression plasmid. By immunizing with the ectodomains of zebrafish cell surface receptor proteins expressed in mammalian cells and screening for formalin-resistant epitopes, we selected antibodies that gave expected staining patterns on wholemount fixed zebrafish embryos.

    Conclusions: This method can be used to quickly select several high quality monoclonal antibodies from a single immunized mouse and facilitates their distribution using plasmids.

    Funded by: NINDS NIH HHS: R01NS063400; Wellcome Trust: 077108/Z/05/Z

    BMC biology 2010;8;76

  • Dopaminergic neuronal cluster size is determined during early forebrain patterning.

    Russek-Blum N, Gutnick A, Nabel-Rosen H, Blechman J, Staudt N, Dorsky RI, Houart C and Levkowitz G

    Department of Molecular Cell Biology, Weizmann Institute of Science, Rehovot 76100, Israel.

    We have explored the effects of robust neural plate patterning signals, such as canonical Wnt, on the differentiation and configuration of neuronal subtypes in the zebrafish diencephalon at single-cell resolution. Surprisingly, perturbation of Wnt signaling did not have an overall effect on the specification of diencephalic fates, but selectively affected the number of dopaminergic (DA) neurons. We identified the DA progenitor zone in the diencephalic anlage of the neural plate using a two-photon-based uncaging method and showed that the number of non-DA neurons derived from this progenitor zone is not altered by Wnt attenuation. Using birthdating analysis, we determined the timing of the last cell division of DA progenitors and revealed that the change in DA cell number following Wnt inhibition is not due to changes in cell cycle exit kinetics. Conditional inhibition of Wnt and of cell proliferation demonstrated that Wnt restricts the number of DA progenitors during a window of plasticity, which occurs at primary neurogenesis. Finally, we demonstrated that Wnt8b is a modulator of DA cell number that acts through the Fz8a (Fzd8a) receptor and its downstream effector Lef1, and which requires the activity of the Fezl (Fezf2) transcription factor for this process. Our data show that the differential response of distinct neuronal populations to the Wnt signal is not a simple interpretation of their relative anteroposterior position. This study also shows, for the first time, that diencephalic DA population size is modulated inside the neural plate much earlier than expected, concomitant with Wnt-mediated regional patterning events.

    Funded by: Medical Research Council: G0400037, G9900989

    Development (Cambridge, England) 2008;135;20;3401-13

  • Otx1l, Otx2 and Irx1b establish and position the ZLI in the diencephalon.

    Scholpp S, Foucher I, Staudt N, Peukert D, Lumsden A and Houart C

    MRC Centre for Developmental Neurobiology, New Hunt's House, Guy's Campus, King's College London, London SE1 1UL, UK. steffen.scholpp@kcl.ac.uk

    The thalamic complex is the major sensory relay station in the vertebrate brain and comprises three developmental subregions: the prethalamus, the thalamus and an intervening boundary region - the zona limitans intrathalamica (ZLI). Shh signalling from the ZLI confers regional identity of the flanking subregions of the ZLI, making it an important local signalling centre for regional differentiation of the diencephalon. However, our understanding of the mechanisms responsible for positioning the ZLI along the neural axis is poor. Here we show that, before ZLI formation, both Otx1l and Otx2 (collectively referred to as Otx1l/2) are expressed in spatially restricted domains. Formation of both the ZLI and the Irx1b-positive thalamus require Otx1l/2; embryos impaired in Otx1l/2 function fail to form these areas, and, instead, the adjacent pretectum and, to a lesser extent, the prethalamus expand into the mis-specified area. Conditional expression of Otx2 in these morphant embryos cell-autonomously rescues the formation of the ZLI at its correct location. Furthermore, absence of thalamic Irx1b expression, in the presence of normal Otx1l/2 function, leads to a substantial caudal broadening of the ZLI by transformation of thalamic precursors. We therefore propose that the ZLI is induced within the competence area established by Otx1l/2, and is posteriorly restricted by Irx1b.

    Funded by: Medical Research Council: G0400037, G0601064

    Development (Cambridge, England) 2007;134;17;3167-76

  • The prethalamus is established during gastrulation and influences diencephalic regionalization.

    Staudt N and Houart C

    Medical Research Council Centre for Developmental Neurobiology, King's College London, London, United Kingdom.

    The vertebrate neural plate contains distinct domains of gene expression, prefiguring the future brain areas. In this study, we draw an extended expression map of the rostral neural plate that reveals discrete domains inside the presumptive posterior forebrain. We show, by fate mapping, that these well-defined cell populations will develop into specific diencephalic regions. To address whether these early subterritories are already committed to restricted identities, we began to analyse the consequences of ablation and transplantation of these specific cell populations. We found that precursors of the prethalamus are already specified and irreplaceable at late gastrula stage, because ablation of these cells results in loss of prethalamic markers. Moreover, when transplanted into the ectopic environment of the presumptive hindbrain, these cells still pursue their prethalamic differentiation program. Finally, transplantation of these precursors, in the rostral-most neural epithelium, induces changes in cell identity in the surrounding host forebrain. This cell-non-autonomous property led us to propose that these committed prethalamic precursors may play an instructive role in the regionalization of the developing diencephalon.

    Funded by: Medical Research Council: G0400037, G0601064; Wellcome Trust: 062893

    PLoS biology 2007;5;4;e69

  • Dickkopf-1 regulates gastrulation movements by coordinated modulation of Wnt/beta catenin and Wnt/PCP activities, through interaction with the Dally-like homolog Knypek.

    Caneparo L, Huang YL, Staudt N, Tada M, Ahrendt R, Kazanskaya O, Niehrs C and Houart C

    Medical Research Council Centre for Developmental Neurobiology, King's College London, SE1 1UL London, UK.

    Dickkopf-1 (Dkk1) is a secreted protein that negatively modulates the Wnt/beta catenin pathway. Lack of Dkk1 function affects head formation in frog and mice, supporting the idea that Dkk1 acts as a "head inducer" during gastrulation. We show here that lack of Dkk1 function accelerates internalization and rostral progression of the mesendoderm and that gain of function slows down both internalization and convergence extension, indicating a novel role for Dkk1 in modulating these movements. The motility phenotype found in the morphants is not observed in embryos in which the Wnt/beta catenin pathway is overactivated, and that dominant-negative Wnt proteins are not able to rescue the gastrulation movement defect induced by absence of Dkk1. These data strongly suggest that Dkk1 is acting in a beta catenin independent fashion when modulating gastrulation movements. We demonstrate that the glypican 4/6 homolog Knypek (Kny) binds to Dkk1 and that they are able to functionally interact in vivo. Moreover, Dkk1 regulation of gastrulation movements is kny dependent. Kny is a component of the Wnt/planar cell polarity (PCP) pathway. We found that indeed Dkk1 is able to activate this pathway in both Xenopus and zebrafish. Furthermore, concomitant alteration of the beta catenin and PCP activities is able to mimic the morphant accelerated cell motility phenotype. Our data therefore indicate that Dkk1 regulates gastrulation movement through interaction with LRP5/6 and Kny and coordinated modulations of Wnt/beta catenin and Wnt/PCP pathways.

    Funded by: Medical Research Council: G0600198, G9900989; Wellcome Trust

    Genes & development 2007;21;4;465-80

  • Mutations of the Drosophila zinc finger-encoding gene vielfältig impair mitotic cell divisions and cause improper chromosome segregation.

    Staudt N, Fellert S, Chung HR, Jäckle H and Vorbrüggen G

    Max-Planck-Institut für biophysikalische Chemie, Abteilung Molekulare Entwicklungsbiologie, 37077 Göttingen, Germany.

    We describe the molecular characterization and function of vielfältig (vfl), a X-chromosomal gene that encodes a nuclear protein with six Krüppel-like C2H2 zinc finger motifs. vfl transcripts are maternally contributed and ubiquitously distributed in eggs and preblastoderm embryos, excluding the germline precursor cells. Zygotically, vfl is expressed strongly in the developing nervous system, the brain, and in other mitotically active tissues. Vfl protein shows dynamic subcellular patterns during the cell cycle. In interphase nuclei, Vfl is associated with chromatin, whereas during mitosis, Vfl separates from chromatin and becomes distributed in a granular pattern in the nucleoplasm. Functional gain-of-function and lack-of-function studies show that vfl activity is necessary for normal mitotic cell divisions. Loss of vfl activity disrupts the pattern of mitotic waves in preblastoderm embryos, elicits asynchronous DNA replication, and causes improper chromosome segregation during mitosis.

    Molecular biology of the cell 2006;17;5;2356-65

  • Gain-of-function screen for genes that affect Drosophila muscle pattern formation.

    Staudt N, Molitor A, Somogyi K, Mata J, Curado S, Eulenberg K, Meise M, Siegmund T, Häder T, Hilfiker A, Brönner G, Ephrussi A, Rørth P, Cohen SM, Fellert S, Chung HR, Piepenburg O, Schäfer U, Jäckle H and Vorbrüggen G

    Max Planck Institut für biophysikalische Chemie, Göttingen, Germany.

    This article reports the production of an EP-element insertion library with more than 3,700 unique target sites within the Drosophila melanogaster genome and its use to systematically identify genes that affect embryonic muscle pattern formation. We designed a UAS/GAL4 system to drive GAL4-responsive expression of the EP-targeted genes in developing apodeme cells to which migrating myotubes finally attach and in an intrasegmental pattern of cells that serve myotubes as a migration substrate on their way towards the apodemes. The results suggest that misexpression of more than 1.5% of the Drosophila genes can interfere with proper myotube guidance and/or muscle attachment. In addition to factors already known to participate in these processes, we identified a number of enzymes that participate in the synthesis or modification of protein carbohydrate side chains and in Ubiquitin modifications and/or the Ubiquitin-dependent degradation of proteins, suggesting that these processes are relevant for muscle pattern formation.

    PLoS genetics 2005;1;4;e55

  • rolling pebbles (rols) is required in Drosophila muscle precursors for recruitment of myoblasts for fusion.

    Rau A, Buttgereit D, Holz A, Fetter R, Doberstein SK, Paululat A, Staudt N, Skeath J, Michelson AM and Renkawitz-Pohl R

    Developmental Biology, Philipps-Universität Marburg, 35032 Marburg, Germany.

    Mutations in the rolling pebbles (rols) gene result in severe defects in myoblast fusion. Muscle precursor cells are correctly determined, but myogenesis does not progress significantly beyond this point because recognition and/or cell adhesion between muscle precursor cells and fusion-competent myoblasts is disturbed. Molecular analysis of the rols genomic region reveals two variant transcripts of rols due to different transcription initiation sites, rols6 and rols7. rols6 mRNA is detectable mainly in the endoderm during differentiation as well as in malpighian tubules and in the epidermis. By contrast, rols7 expression is restricted to the mesoderm and later to progenitor descendants during somatic and pharyngeal muscle development. Transcription starts at the extended germ band stage when progenitor/founder cells are determined and persists until stage 13. The proteins encoded by the rols gene are 1670 (Rols6) and 1900 (Rols7) amino acids in length. Both forms contain an N-terminal RING-finger motif, nine ankyrin repeats and a TPR repeat eventually overlaid by a coiled-coil domain. The longer protein, Rols7, is characterized by 309 unique N-terminal amino acids, while Rols6 is distinguishable by 79 N-terminal amino acids. Expression of rols7 in muscle founder cells indicates a function of Rols7 in these cells. Transplantation assays of rols mutant mesodermal cells into wild-type embryos show that Rols is required in muscle precursor cells and is essential to recruit fusion-competent myoblasts for myotube formation.

    Development (Cambridge, England) 2001;128;24;5061-73

Zenon Zenonos

- Postdoctoral Fellow

I graduated the Department of Biology, University of Patras (Greece)in 2007. Graduating as the valedictorian of my class, I received the Alexander S. Onassis Distinguished Scholar Award, and I joined Dr. Flytzanis lab, where I pursued a two year research, towards an MSc degree in Biotechnology. With Dr. Flytzanis, I investigated the role of COUP-TF in the development of sea urchin Paracentrotus lividus. In 2009 I joined the WTSI, for a four year PhD programme in Molecular Biology. At Sanger, I worked for two years in the field of induced pluripotency before I join Gavin's lab in 2011.

Research

My research focuses on the elucidation of the protein-protein interactions that take place on the cell surface, during the invasion of erythrocytes by the malaria parasite Plasmodium falciparum. We make use of a mammalian expressing system to produce cell surface protein libraries from both the parasite and erythrocytes. We employ powerful assays, that were developed in the lab, to systematically screen the libraries against each other with the aim to identify novel ligand-receptor pairs. The hits are then followed in vivo. Another aspect of my research involves the development of therapeutics based on the ligand-receptor pairs identified in the lab.

References

  • Rapid and efficient reprogramming of somatic cells to induced pluripotent stem cells by retinoic acid receptor gamma and liver receptor homolog 1.

    Wang W, Yang J, Liu H, Lu D, Chen X, Zenonos Z, Campos LS, Rad R, Guo G, Zhang S, Bradley A and Liu P

    Wellcome Trust Sanger Institute, Hinxton CB10 1HH, United Kingdom.

    Somatic cells can be reprogrammed to induced pluripotent stem cells (iPSCs) by expressing four transcription factors: Oct4, Sox2, Klf4, and c-Myc. Here we report that enhancing RA signaling by expressing RA receptors (RARs) or by RA agonists profoundly promoted reprogramming, but inhibiting it using a RAR-α dominant-negative form completely blocked it. Coexpressing Rarg (RAR-γ) and Lrh-1 (liver receptor homologue 1; Nr5a2) with the four factors greatly accelerated reprogramming so that reprogramming of mouse embryonic fibroblast cells to ground-state iPSCs requires only 4 d induction of these six factors. The six-factor combination readily reprogrammed primary human neonatal and adult fibroblast cells to exogenous factor-independent iPSCs, which resembled ground-state mouse ES cells in growth properties, gene expression, and signaling dependency. Our findings demonstrate that signaling through RARs has critical roles in molecular reprogramming and that the synergistic interaction between Rarg and Lrh1 directs reprogramming toward ground-state pluripotency. The human iPSCs described here should facilitate functional analysis of the human genome.

    Funded by: Medical Research Council: G0700665; Wellcome Trust: 077186/Z/05/Z

    Proceedings of the National Academy of Sciences of the United States of America 2011;108;45;18283-8

* quick link - http://q.sanger.ac.uk/cellsurf