2010 Seminars

Christmas Special Seminar. December 15@ noon
Systems biology analysis and modeling of mRNA, protein and turnover in a bacterium
Dr Luis Serrano.
CRG, Barcelona

Summary: Biological function and cellular responses to environmental perturbations are regulated by a complex interplay of DNA, RNA and proteins inside cells. To understand these central processes in living systems at the molecular level, we integrated genome-wide experimental data of mRNA, absolute protein abundances and individual half-lives. We provide a fine-grained, fully quantitative and dynamic picture of the inner life of a cell under various external conditions in the genome-reduced bacterium Mycoplasma pneumoniae. Proteome dynamics in response to cellular perturbations reveal specific stress response strategies. Translation efficiency predominantly regulates mRNA-protein ratios. Stochastic simulations show how low translation efficiency and long protein half-lives effectively reduce biological noise. Protein abundances reflect protein complex dynamics, are relatively conserved at pathway level and reflect lifestyles of organisms. Our study provides the most detailed and complete integrative analysis to date of absolute quantities and the dynamic interplay of mRNA and proteins, the central biomolecules of a cell.
CABD Seminar December 10@ 10:30
In vitro prion replication. Infectivity, strain and transmission barrier phenomena
Dr. Joaquin Castilla.
Biogune, Derio

Summary: One of the characteristics of prions is their ability to infect some species and not others. This phenomenon is known as the transmission barrier. In general, the transmission barrier is expressed by an incomplete attack rate and long incubation times which become shorter after serial inoculation passages. The absence of natural TSE cases and/or failed experimental transmissions has suggested that some species could be resistant for prion diseases. Unfortunately, the molecular basis of the transmission barrier phenomenon is currently unknown and we cannot predict the degree of a species barrier simply by comparing the prion proteins from two species. We have conducted a series of experiments using the Protein Misfolding Cyclic Amplification (PMCA) technique that mimics in vitro some of the fundamental steps involved in prion replication in vivo albeit with accelerated kinetics. We have used this method to efficiently replicate a variety of prion strains from, among others, mice, hamsters, bank voles, deer, cattle, sheep and humans. The in vitro generated PrPres possess key prion features, i.e. they are infectious in vivo and maintain their strain specificity. We are using the PMCA to generate infectious PrPres from species hitherto considered to be resistant to prion disease and assay the role that certain amino acids play in the transmission barriers. The correlation between in vivo data and our in vitro results suggest that PMCA is a valuable tool for studying the strength of the transmission barriers between diverse species and for evaluating the potential risks of the newly generated prion species to humans and animals
CABD Seminar December 3@noon
Genesis of the nematode fluid absorption/filtration system
Dr. Michel Labouesse
IGBMC. Illkirch, France

Summary: All living metazoans have evolved a fluid drainage system which removes excess interstitial fluid and/or filters it. In vertebrates, this function is ensured by the kidney and lymphatic vessels. I will describe the nematode equivalent organ, called the excretory system. This organ is composed of five cells, one of which, the excretory cell, ensures the filtration function. By characterizing mutants defective for excretory cell formation we have uncovered steps for excretory cell genesis, from its early determination to lumen formation. Interestingly, the vertebrate homologue of a gene at the top of the hierarchy for excretory cell formation is also essential for lymphatic vessel determination, suggesting an ancient evolutionary origin. We believe that our identification of its potential targets should help understand lymphatic capillary formation. I will also present the CLEM method we have developped to characterize the phenotypes of our mutants.
CABD Seminar November 26@noon
Cell wars: cell competition via growth factor signalling
Dr. Eugenia Piddini
The Wellcome Trust/Cancer Research. Cambridge (UK).

Summary: Deregulation of Wnt signalling is often associated with cancer. Wingless, a Drosophila Wnt homolog, is an important regulator of cell fate specification, survival and proliferation. We have recently found that, in Drosophila, cells experiencing different Wingless signalling levels engage in competitive cell interactions. For example, local autonomous over-activation of Wg signalling (resulting from mutations in APC or axin) leads to apoptosis in surrounding normal cells. This process appears to be distinct from classical cell competition as it does not involve Myc. We find that instead it requires Notum, a conserved secreted negative feedback inhibitor of Wnt signalling. By amplifying local differences in Wingless signaling, Notum may serve as an early trigger of Wingless signaling-dependent competition. We propose that analogous cell interactions could provide a competitive advantage to Wnt signaling-dependent precancerous cells in humans.
CABD Seminar November 22@noon
Environmental stress alters developmental regulation of serotonin levels in Caenorhabditis elegans
Dr. Miguel Estevez
Department of Neurology, University of Arizona, USA.

Summary: Stress during early neurodevelopment is thought to have long-lasting repercussions on mood in humans. Given the importance of Serotonin in the regulation of mood it has been hypothesized that regulation of the levels of this neurotransmitter may play a role. However, the neurodevelopmental signaling pathways that connect environmental stress to serotonin remain poorly defined in vertebrates. Using the genetic model C. elegans we have defined genetic pathways that translate the environmental stress of elevated temperature into transcriptional regulation of the rate-limiting enzyme for serotonin, tryptophan hydroxylase. In this model of environmental regulation of mood Calcium channel signaling through a Cam Kinase II dependent pathway interacts with CREB signaling to determine the level of tryptophan hydroxylase in adults exposed to noxious temperature stimuli as larvae.
CABD Seminar November 19@noon
Beyond extracellular matrix degradation: New functions of MT-MMPs in cellular biology
Dr. Alicia García Arroyo
CNIC, Madrid.

Summary: Matrix metalloproteinases (MMPs) are endopeptidases in charge of degrading extracellular matrix components but also of modulating the bioactivity of transmembrane receptors and soluble factors. Since we are interested in biological processes related to cell motility and invasion in the angiogenesis and inflammatory contexts, we have focused our research in members of the MMP family in particular in MT1-MMP and MT4-MMP. These two MMPs are anchored to the plasma membrane by a transmembrane domain and a GPI link, respectively, what make them especially suitable for pericellular proteolysis and modulation of cell behaviour. By combining proteomic approaches and functional analysis at the cellular and whole organism levels we have expanded the functions of these two MT-MMPs in cells that are instrumental to the inflammatory response. Thus, by using SILAC we have identified the degradome (collection of substrates) of MT1-MMP in endothelial and myeloid cells; these new data point to expected but also unexpected functions for MT1-MMP in pathophysiology. In addition, we have recently characterized in detail a novel function of MT1-MMP in regulating macrophage motility along the process of cellular fusion that takes place during osteoclast or inflammatory giant cell formation (Gonzalo et al., Dev. Cell, 2010). Notably, the catalytic activity of MT1-MMP is dispensable for this function; instead, MT1-MMP cytosolic tail is required in macrophages for optimal Rac1 activity through the binding to the adaptor protein p130Cas. Novel insights about the cellular functions of the poorly studied protease MT4-MMP will also be discussed. Our findings indicate that MT-MMPs contribution to cellular biology is likely dependent on the cellular context and it is beyond their active role in degrading the extracellular matrix.
CABD Seminar November 5@noon
DIFase another piece of the Dictyostelium discoideum development puzzle.
Dr. Francisco Velázquez Duarte
MRC-Laboratory of Molecular Biology, Cambridge, UK

Summary: D. discoideum, the social amoeba, triggers a very simple developmental program upon starvation. This program involves the gathering of pre-existing starving cells and their differentiation into either spore cells -which are a resisting form - or stalk cells, that will form ancillary structures to facilitate the spores dispersion and survival. DIF, a chlorinated alkyl-phenone has been reported to be a major stalk cell inducer, synthesized by pre-spores cells, and degraded by stalk cells. How this molecule acts during development is still far from understood; here we report the identification and initial characterization of DIF 3,5 dechlorinase, DIFase the first enzyme in DIF degradation.
CABD Seminar October 29@noon
Beyond classical genetics: how to shed light on complex traits and diseases by integrating mouse diversity, non-mendelian and systems genetics
Dr. Elena de la Casa
Centro Regional de Investigaciones Biomédicas Complejo Facultad de Medicina Universidad de Castilla-La Mancha/Parque Científico y Tecnológico de Albacete

Summary: Classical mendelian genetics does not provide a paradigm able to fully integrate genomic and systems genetics studies. In fact, phenomena that defy Mendel’s laws –such as monoallelic and imprinted gene expression- play crucial roles in the inheritance and the phenotypic manifestations of many complex traits and diseases, and are often mediated by epigenetic modifications. Over the years, my research has focused on these phenomena. Although our initial analyses and mapping studies were performed on classical mouse inbreed strains, we moved on to employ wild-derived inbred strains, whose genetic and phenotypic diversity substantially expanded our findings. Ours and others' studies on mouse genetic diversity have set up the basis for a new trend in mouse genetics. I will illustrate this trend in a systems genetics project, and will discuss the implications of a novel genome-wide analysis of DNA methylation and imprinting in this and future studies.
CABD Seminar September 24@noon
Mouse models to dissect cohesin function
Dr. Ana Losada
CNIO, Madrid

Summary: Cohesin is a protein complex that mediates sister chromatid cohesion, a process essential for accurate chromosome segregation. It has a ring-like structure comprised of four subunits: Smc1, Smc3, Rad21/Scc1 and SA/Scc3. Two additional proteins, Pds5 and Wapl, interact closely with cohesin and modulate its behavior. Cohesin is thought to act as a topological linker that embraces the two DNA molecules. Recent evidence suggests that this ability serves also to stabilize chromosomal cis-interactions that contribute to regulate gene expression and DNA replication in interphase cells. Heterozygous mutations in genes encoding cohesin and its chromatin loading factor, Nipbl/Scc2, are responsible for the Cornelia de Lange syndrome. This developmental disorder affects 1:30,000 newborns and is characterized by growth and cognitive retardation. Mutations in cohesin subunits have also been found in human colorectal cancer samples. In order to dissect the different functions of cohesin, how they are regulated and its physiological significance, we have generated mice deficient for cohesin and its regulators.
CABD Seminar September 17@noon
La investigación en Ciencias Marinas y Limnología en la Universidad de Costa Rica
Dr. Álvaro Morales Ramírez (Director CIMAR, UCR)
CIMAR, Universidad de Costa Rica

Summary: Creado en 1979, el Centro de Investigación en Ciencias del Mar y Limnlogía (CIMAR) de la Universidad de Costa Rica es el único centro de investigación en este campo en el país. El CIMAR ha desarrollado más de 270 proyectos en 11 áreas de investigación, incluyendo cambio global, manejo costero integrado, biotecnología de algas, contaminación costera, recursos pesqueros y diversidad acuática. Los esfuerzos de investigación se realizan en la costas Pacífica y Caribe, el Parque Nacional Isla del Coco, la Reserva Natural Isla del Caño, y lagos, lagunas y ríos de todo el país. El CIMAR apoya los Programas de Maestría en Biología y de Maestría en Gestión Integrada de Áreas Costeras Tropicales (dentro del marco del programa Alfa (América Latina-Formación Académica) de la Unión Europea) y el Programa de Doctorado en Ciencias de la UCR. En total se han realizado 108 másters y tesis, de las cuales el 40,7% corresponde a estudiantes extranjeros, especialmente centroamericanos. La cooperación internacional ha sido fundamental en el desarrollo del Centro con colaboradores en instituciones estadounidenses, europeas y de América Latina. Nuevas áreas emergentes con gran potencial como la Genética de Organismos Marinos, la Microbiología y Biogeoquímica Estuarina, la Bioprospección Marina y la Diversidad de Aguas Profundas permitirán la integración del trabajo con nuevos Centros y/o Institutos de Investigación dentro y fuera del país.
CABD Seminar September 15@noon
Finding the cell middle
Dr. Fred Chang
Columbia University, New York

Summary: I will discuss how cells divide in the middle. During this process, cells probe their own cell shape and dimensions using microtubules to position the nucleus or spindle. These organelles then help to position of the cell division plane for cytokinesis. How do cells sense their own shape? What are some of the signals that position the cell division plane? I will present our studies in spatial regulation in two different model organisms: fission yeast and sea urchin.
CABD Seminar June 28@noon
Processes of gene activation and repression, involved in the initiation of thyroid and liver development
Dr. Pablo Recacha
Instituto de Investigaciones Biomédicas (IIBM), CSIC-UAM

Summary: The development of endoderm derived organs is a very interesting model to study the mechanisms of action of transcriptional activators and repressors in vivo. In this thesis, we have focused on such mechanisms, studying different processes that take place during thyroid and liver development. The specification of a group of cells of the endoderm to a thyroid fate, begins with the expression of Titf1, FoxE1 and Pax8 transcription factors. Although, signals that regulate this onset have not been reported it has been proposed that these signals could be common to signals that regulate other organs of the same embryonic origin. In this work, we have identify Sonic Hedgehog pathway (Shh) as one of these signals responsible for the thyroid specification, demonstrating that this pathway regulates Titf1, FoxE1 and Pax8 expression by direct binding of Gli transcription factors to the promoter region of those genes. The main role of Shh pathway is played over FoxE1, a forkhead transcription factor with has the ability to open compacted chromatin and that is responsible for the onset of thyroglobulin and thyroperoxidase expression, two important thyroid differentiation markers. In the embryonic development of this gland, it has been reported a delay between the expression of thyroid transcription factors and their target genes. In order to study this process, we have used the hepatic organogenesis as a experimental model, because the development of the liver is almost completely understood. The endodermal cells which contribute to the hepatic bud begin its differentiation with the expression of FoxA1 and FoxA2 transcription factors and 6 hours later, they promote the expression of their target genes, Albumin and Transthyretin. We demonstrate that the mechanism responsible for this delay of liver specific genes, is the temporally expression of Groucho transcriptional correpresors during this period. These factors, repress the activation function of FoxA1 and FoxA2 on Albumin and Transthyretin enhancers.
CABD Seminar June 25@noon
Nestin+ MSCs: self-renewing stem cells with HSC niche functions
Dr. Simón Méndez-Ferrer
Mount Sinai School of Medicine, New York, USA

Summary: The identity of the cells forming the haematopoietic stem cell (HSC) niche remains unclear. Bone-lining osteoblasts and preosteoblasts, osteoclasts, megakaryocytes, endothelial and reticular cells, as well as mesenchymal progenitors have been proposed to contribute to the HSC niche. However, despite their broad therapeutic potential, mesenchymal stem cells (MSCs) and their putative functions in the HSC niche remain poorly defined due to MSCs heterogeneity, the inability to assess their in vivo self-renewal and the paucity of specific markers that allow for their identification, isolation and genetic manipulation. Previous work from the Frenette lab has shown that HSC mobilization induced by granulocyte colony-stimulating factor (G-CSF) requires signals from the sympathetic nervous system and is associated with inhibition of bone-lining osteoblasts. However, physiological release of HSCs into the bloodstream follows circadian oscillations of Cxcl12/Sdf-1 expression triggered by cyclical activation of the _3-adrenergic receptor, which is not expressed by osteoblasts but other stromal elements (Mendez-Ferrer et al. 2008; Nature 452:442-7). We have recently identified these stromal cells as bona fide MSCs identified using regulatory elements of the intermediate filament protein nestin. In the bone marrow of Nestin-Gfp transgenic mice, CD31- CD45- GFP+ peri-vascular cells expressing nestin are anatomically associated with HSCs and sympathetic fibres. They are highly enriched in the expression of HSC maintenance genes, which are selectively downregulated in this cell population following G-CSF treatment or _3-adrenergic receptor stimulation. Although they represent a small subset of stromal cells, Nestin:GFP+ cells contain all the colony-forming unit-fibroblastic (CFU-F) activity and have the exclusive capacity within bone marrow cells of forming multipotent clonal spheres able to self-renew, differentiate into mesenchymal lineages and transfer haematopoietic activity in serial transplantations using heterotypic bone ossicles. The proliferation and osteoblastic differentiation of Nestin:GFP+ cells is regulated by cytokines (G-CSF), neural (sympathetic) and hormonal (PTH) input. Lineage-tracing studies in Nes-Cre/R26R mice have shown the contribution of nestin+ cells in endochondral and membranous ossification. Administration of tamoxifen to adult Nes-CreERT2 mice bred to a reporter line has revealed the presence of GFP+ osteolineage cells after 8-month chasing, suggesting an active role for adult nestin+ MSCs in physiological bone turnover. In vivo nestin+ cell depletion using Nes-CreERT2 mice bred to a Cre-inducible diphtheria toxin receptor line rapidly reduces HSC content in the bone marrow, owing at least partially to their mobilization to extramedullary sites. Purified HSCs injected into lethally irradiated mice rapidly home near bone marrow nestin+ MSCs, whereas in vivo nestin+ cell depletion significantly reduces bone marrow homing of haematopoietic progenitors. These results indicate that nestin regulatory elements may represent a valuable tool to prospectively isolate and genetically manipulate MSCs. They also uncover a close relationship between two different somatic stem cells and reveal essential functions for MSCs in the HSC niche.
CABD Seminar June 11@noon
Chipping away at the regulatory architecture of the genome
Dr. Rob White
Physiology, Development and Neuroscience, University of Cambridge

Summary: How is the regulation of gene expression facilitated and supported by the overall architecture of the genome? Insulators, blocking inappropriate enhancer action and bounding chromatin domains, are key elements of genome architecture. I will present our studies on the association of insulators with the regulation of Hox gene expression in the Bithorax complex in Drosophila. I will also present our recent work on Hox targets and discuss the role of genome architecture/chromatin accessibility on target gene selection by transcription factors.
CABD Seminar June 4@noon
Wnt, differentiation and apoptosis in C.elegans
Dr. Juan Cabello
Centre for Biomedical Research of La Rioja - CIBIR, Logroño

Summary: During development, the processes of cell division, differentiation and apoptosis must be precisely coordinated in order to maintain tissue homeostasis. The nematode C.elegans is a powerful model system to study cell death and its control. C.elegans apoptotic cells condense and form refractile corpses under differential interference contrast (DIC) microscopy. Activation of the GTPase CED-10/Rac in a neighbouring cell mediates the recognition and engulfment of the cell corpse. This activation of CED-10/Rac is carried out by the Wnt pathway among other pathways. After inclusion of the engulfed corpse in a phagosome, different proteins are sequentially recruited onto this organelle to promote its acidification and fusion with lysosomes, leading to the enzymatic degradation of the cell corpse. We show that CCZ-1, a protein conserved from yeasts to humans, mediates the digestion of the apoptotic corpses. We also provide new insights in the regulation of the Wnt pathway.
CABD Seminar May 10@noon
Genes, screens and drugs, and what they tell us about formation and regeneration of the zebrafish lateral line
Dr. Miguel Allende
Universidad de Chile, Santiago de Chile

Summary: evelopment of the lateral line system in zebrafish is remarkable as it combines features such as coordinated collective migration that is simultaneous with cell division, cell specification and differentiation of a functional sensory organ, all in a matter of hours. The neuromasts, individual sensory patches distributed over the body of the fish, contain sensory hair cells, which are able to regenerate when destroyed by trauma or toxicity. In adult fish, entire regions of the lateral line system and the axons that innervate the sensory cells can regenerate as well, after amputation of the tail, for example. We have undertaken the task of identifying genes that are involved in the developmental and regenerative events that take place in this system. New genes expresse in the progenitor cells have been found through a combination of cell sorting and microarray analysis. We show that some of these possess roles in progenitor cell migration and sensory cell differentiation. Other genes have been identified through genetic screens and candidate gene analysis. Several of these have become useful markers for the different cell types present in the lateral line system. We have also described novel events that occur during lateral line system regeneration. Axotomy of the lateral line nerve is followed by Wallerian degeneration of the distal portion of the axon, but is followed rapidly by growth of new axons. We have observed that correct pathfinding by the newly growing axons requires functional hair cells as targets for innervation. These experiments show that, while survival of hair cells and neurons are independent of each other, regeneration of the lateral line nerve is partially dependent on an intact set of sensory cells. Secondly, we show that all lateral line components can reform over regenerated tail fin tissue. This process requires de novo migration of individual progenitor cells from existing neuromasts. Finally, we have recently discovered that damage to hair cells and other lateral line components elicits a potent innate immune inflammatory response. Specific migration of neutrophils and macrophages to damaged neuromasts has been used to develop an assay to detect anti-inflammatory activity of small molecules, thus offering an attractive alternative for drug screens.
CABD Seminar May 7 @noon
Chinmo is a functional effector of the JAK/STAT pathway that mediates eye development, tumorigenesis and stem cell self-renewal in Drosophila.
Dr. Erika Bach
NYU, New York, USA
CABD Seminar May 7 @10am
Dr. Ian Holt
MRC-Mitochondrial Biology Unit, Cambridge, UK
CABD Seminar April 16 @1pm
An RNAi-based screen identifies the nucleoporin NPP-3 as a novel regulator of mitotic onset in C. elegans embryos
Dr. Virginie Hamelhachet
ISREC-Swiss Institute for Experimental Cancer Research. Lausanne Switzerland

Summary: Mitosis is a tightly regulated process that ensures faithful partition of the genetic material to daughter cells. We set out to investigate the nature of spatial and temporal cues controlling mitotic entry using one cell-stage Caenorhabditis elegans embryos. Previously, we had developed a sensitive in vivo assay for mitotic entry, in which this process is asynchronous when the male and female pronuclei are separated. Using this assay, we had established that centrosomes dictate the onset of mitosis and that the Aurora-A kinase AIR-1 is necessary for timely entry into mitosis. In order to decipher the mechanisms by which centrosomes and AIR-1 regulate mitotic entry, we conducted a visual RNAi-based screen using the sensitiv in vivo assay that we developed on 450 selected candidate genes. Interestingly, we identified a subset of nucleoporins as potential modulators of mitosis. In particular, we established that RNAi-mediated depletion of NPP-3 basically abrogates the asynchrony between the male and female pronuclei in our assay. Together, these findings indicate that NPP-3 regulates timely mitotic entry. We next raised antibodies against NPP-3 and, strikingly, found that NPP-3 is removed locally from the nuclear envelope in the vicinity of centrosomes prior to mitosis onset. We demonstrated that this local removal is independent of dynein or microtubules, but requires the presence of centrosomes and the function of AIR-1.
CABD Seminar April 26 @noon
Structures and functions of mitotic chromosomes
Dr. Alexander Strunnikov
Laboratory of Immunopathology. National Institute of Allergy and Infectious Diseases. National Institutes of Health

Summary: Recent advances in cancer genomics prove that tumorigenesis correlates with massive chromosomal rearrangements and other types of pervasive genome instability. Therefore, studying mechanisms that ensure high-fidelity chromosome segregation and the ensuing genome integrity is of paramount importance both for understanding biology of complex diseases and for finding cures. Since our discovery of SMC (Structural Maintenance of Chromosomes) proteins 20 years ago, our work on SMC complexes cohesin and condensin, topoisomerase II, SUMO, and other key chromosomal components has confirmed that the higher order structure of mitotic chromosomes in eukaryotes possesses its own unique function - to ensure that sister chromatids segregate properly in anaphase, that is when major cell cycle checkpoint systems are either turned off or become inadequate. Thus, the core direction in our research is to understand the “gene-drivers” of genome instability, with respect to (a) their functional, mechanistic and pathway identity, (b) the specific forms of chromosome instability generated upon inactivation of a specific pathway, (c) the allelic state of these genes in somatic tumors, as putative targets for anti-cancer treatments. Such an approach is exemplified by our most recent study on Condensins I and II that are essential ATP-dependent complexes necessary for chromosome condensation and segregation in mitosis. While investigating the structural role of human condensins in centromeres and kinetochores we demonstrated that significant chromosome damage induced by the loss of condensins was not properly detected by checkpoints, thus resulting in potentially heritable genome instability. Furthermore, the depletion of human condensin activity results in a significant loss of loading of CENP-A (the histone H3 variant) - the epigenetic mark of centromere identity. While loss of condensin and CENP-A transiently activates the mitotic checkpoint, it nevertheless does not prevent chromosome missegregation. This inability of the functional mitotic checkpoint to correct kinetochore misattachments is due to condensin-depletion-dependent deformation of both inner kinetochores and the microtubule-capturing module, resulting in kinetochore separation from the Aurora B pool and in ensuing reduced kinase activity at centromeres. Moreover, our results show that the dysfunction of Aurora B is the key defect leading to chromosome missegregation in condensin-depleted cells. The ongoing genome-wide characterization of instability zones that emerge specifically in condensin-depleted cells by ChIP-seq and ChIP-chip indicates that the affected regions include fragile sites, SNP hotspots, and oligonucleotide expansion/variability sites. While some of these locations fall within known genome instability zones, many are novel, probably condensin loss-specific. This approach exemplifies the research pipeline that is to be followed with other chromosomal proteins: to investigate the pathway-specific components of genome instability genome-wide. The specific focus is on the genes that, when mutated/dysfunctional, could in “one hit” both generate genome instability and override checkpoints. The additional strength of our current approach is the inclusion of the repeated part of human genome, particularly tandem repeat zones, to achieve truly genome-wide analysis. The second direction on our work, systemic structure-function analysis of mitotic chromatin, was prompted by the critical mass of data indicating that chromosome proteins form mitosis-specific interactions with each other and with DNA. The molecular nature of such interactions in mitotic chromosomes is obscure. Therefore, we established an experimental system for genome-wide studies on structural components of mitotic chromatin. We aimed to solve three particular problems: (a) functional specialization of condensin I and condensin II in vertebrates; (b) the structural identity of inner centromere; (c) the mitotic role of CTCF. Both strategic directions of our research, i.e. (1) structure-function and pathway analysis of “gene-drivers” in genome instability and (2) systemic integrative approach to solving the nature of mitotic chromatin, are relevant to the biological problems of genome instability, cancer, and potentially of cell fate reprogramming in general.
CABD Seminar April 16 @1pm
An RNAi-based screen identifies the nucleoporin NPP-3 as a novel regulator of mitotic onset in C. elegans embryos
Dr. Virginie Hamelhachet
ISREC-Swiss Institute for Experimental Cancer Research. Lausanne Switzerland

Summary: Mitosis is a tightly regulated process that ensures faithful partition of the genetic material to daughter cells. We set out to investigate the nature of spatial and temporal cues controlling mitotic entry using one cell-stage Caenorhabditis elegans embryos. Previously, we had developed a sensitive in vivo assay for mitotic entry, in which this process is asynchronous when the male and female pronuclei are separated. Using this assay, we had established that centrosomes dictate the onset of mitosis and that the Aurora-A kinase AIR-1 is necessary for timely entry into mitosis. In order to decipher the mechanisms by which centrosomes and AIR-1 regulate mitotic entry, we conducted a visual RNAi-based screen using the sensitiv in vivo assay that we developed on 450 selected candidate genes. Interestingly, we identified a subset of nucleoporins as potential modulators of mitosis. In particular, we established that RNAi-mediated depletion of NPP-3 basically abrogates the asynchrony between the male and female pronuclei in our assay. Together, these findings indicate that NPP-3 regulates timely mitotic entry. We next raised antibodies against NPP-3 and, strikingly, found that NPP-3 is removed locally from the nuclear envelope in the vicinity of centrosomes prior to mitosis onset. We demonstrated that this local removal is independent of dynein or microtubules, but requires the presence of centrosomes and the function of AIR-1.
CABD Seminar April 16 @ 10am
- Howart Jacobs (Institute of Medical Technology - Tampere University, Finland)
Mitochondrial disease in flies

- Rafael Garesse (Instituto de Investigaciones Biomédicas "Alberto Sols", CSIC-UAM, Madrid)
Modelling OXPHOS defects in flies
CABD Seminar April 9 @noon
Fast regulation of AP-1 activity through interaction of lamin A/C, ERK1/2 and c-Fos at the nuclear envelope
Dr. Vicente Andrés
Spanish National Cardiovascular Research Center (CNIC), Madrid

Summary: A-type lamins (lamins A and C), encoded by the LMNA gene, are major protein constituents of the mammalian nuclear lamina, a complex structure of the nuclear envelope (NE) that acts as a scaffold for protein complexes that regulate nuclear structure and functions. Interest in these proteins has increased in recent years with the discovery that LMNA mutations cause a variety of human diseases termed laminopathies, including progeroid syndromes and disorders that primarily affect striated muscle, adipose, bone, and neuronal tissues. Evidence is accumulating that lamin A/C and associated NE proteins regulate gene expression in health and disease through interplay with signal transduction pathways, transcription factors, and chromatin-associated proteins. W have demonstrated that A-type lamins regulate AP-1 transcription factor activity by sequestering c-Fos at the NE in an ERK1/2-dependent manner. We find that c-Fos and lamin A/C interact in vitro and in vivo through leucine residues and that these proteins colocalize at the NE in starvation-synchronized quiescent cells, which lack detectable AP-1 DNA-binding activity. Serum-induced up-regulation of AP-1 DNA-binding activity coincides with c-Fos nucleoplasmic accumulation, and serum stimulation rapidly releases preexisting c-Fos from the NE via ERK1/2-dependent phosphorylation, thus leading to fast AP-1 activation in advance of de novo c-Fos synthesis. Moreover, ERK1/2 interacts with lamin A/C and colocalizes with c-Fos and A-type lamins at the NE, and lamin A/C overexpression impairs the formation of active c-Fos/c-Jun heterodimers, inhibits AP-1–dependent DNA-binding activity and transcription, and causes a growth arrest that can be partially rescued by c-Fos overexpression. Conversely, LMNA-null cells exh bit scant perinuclear c-Fos localization, increased AP-1 DNA-binding and transcriptional activity, and enhanced proliferation. NE-bound ERK1/2 may therefore function as a molecular switch for rapid mitogen-dependent AP-1 activation through phosphorylation-induced release of preexisting c-Fos from its inhibitory interaction with lamin A/C. Future studies should address whether other signal transducers and AP-1 family members are regulated by interactions with A-type lamins and other NE proteins and whether disease-causing lamin A/C mutations affect the c-Fos–ERK1/2 interplay at the NE with consequent changes in AP-1 activity and cell cycle progression.
CABD Seminar April 5 @noon
Microglia shape adult hippocampal neurogenesis through apoptosis-coupled phagocytosis
Dr. Amanda Sierra
Laboratory of Neuroendocrinology, Rockefeller University, New York, USA

Summary: In the adult hippocampus, neuroprogenitor cells in the subgranular zone (SGZ) of the dentate gyrus give rise to newborn neuroblasts. However, only a small subset these cells integrate into the hippocampal circuitry as mature neurons at the end of a four-week period. Here, we show that the majority of the newborn cells undergo death by apoptosis in the first one to four days of life, during the transition from amplifying neuroprogenitors to neuroblasts. These apoptotic newborn cells are rapidly cleared out through phagocytosis by unchallenged microglia present in the adult SGZ niche. Phagocytosis by the microglia is efficient and undeterred by aging or inflammatory challenge. Our results suggest that the critical period of newborn cell survival occurs within a few days of birth and reveal a new role for microglia in maintaining the homeostasis of the baseline neurogenic cascade.
CABD Seminar March 12 @noon
Haematopoietic Stem Cell Emergence during Mouse Development
Dr. Katrin Ottersbach
Department of Haematology,Cambridge Institute for Medical Research, University of Cambridge, ENGLAND UK

Summary: The first adult-type haematopoietic stem cells (HSCs) are detected at embryonic day (E) 10.5 of mouse development in the AGM region, which comprises the dorsal aorta and the developing gonads and mesonephros. We have recently carried out a spatial and temporal comparative gene expression analysis of the AGM to identify novel regulators of HSC emergence. Our functional validation experiments identified regulators of various aspects of HSC behaviour, including expansion and migration, as well as negative regulators. Furthermore, upon functional classification of the differentially expressed genes, it was noted that, in addition to haematopoiesis-associated regulators, many genes involved in the development of the tissues surrounding the dorsal aorta are also upregulated at the time of HSC emergence, suggesting that HSC generation occurs in coordination with the development of other organs. Our investigations into the role that Gata3, one of the upregulated genes, plays in HSC production confirmed that this is indeed the case. We have determined that Gata3 controls HSC generation via its role in the sympathetic nervous system, thus functionally linking these two developing systems. However, preliminary experiments suggest that Gata3 may also play a more direct role in the immediate HSC microenvironment. Future studies will concentrate on the identification of Gata3 target genes in this context. Other lines of work focus on identifying cytokine signalling pathways involved in HSC production and finding functional connections between the candidate regulators identified so far and how miRNAs contribute to this network of HSC regulators.
CABD Seminar March 5 @ noon 
SoxD genes in the control of vertebrate neurogenesis
Dr. Aixa Morales
Dpt. Cellular, Molecular and Developmental Neurobiology
Instituto Cajal (CSIC)

Summary: We are interested in getting an integrated view of the coordination of processes such as cell cycle exit/progression, control of proneural program and cell-type specification and differentiation during the course of central nervous system development in vertebrates. We use as an entry point the functional analysis of the SoxD family of transcription factors in the context of spinal cord development and their possible interactions with well known signalling pathways such as the Wnt and Shh pathways. We have shown that Sox5 is expressed in neural progenitors in the spinal cord, and in a subpopulation of dorsal dI3 interneurons. Through gain- and loss-of-function analyses, we found that Sox5 controls the timing of cell cycle exit in neural progenitors at the G1-S transition by counteracting the mitotic effect of the Wnt/_-catenin pathway. Mechanistically, by increasing the transcriptional levels of the negative regulator Axin2, Sox5 would control the feedback repressor pathway regulating Wnt signalling. Furthermore, we have found that Sox5 downregulation in postmitotic cells is necessary for the progression of the differentiation program and that Sox5 is essential for the survival of dI3 interneurons. Hence, these data situate Sox5 as an important brake of Wnt/_-catenin mitogenic activity during the progression of neurogenesis. Furthermore, Sox5 regionalized pattern of expression along the dorso-ventral axis is important for the correct specification of dorsal as well as ventral subtypes of interneuron precursors, contributing to the coordination of cell cycle exit and cell fate-specification.
CABD Seminar February 19 @ noon 
Regulon-specific control of transcription elongation across the yeast genome
Dr. Sebastián Chávez
Departamento de Genetica, Universidad de Sevilla

Abstract: Transcription elongation by RNA polymerase II was often considered an invariant non-regulated process. However, genome-wide studies have shown that transcriptional pausing during elongation is a frequent phenomenon in tightly-regulated metazoan genes. Using a combination of ChIP-on-chip and genomic run-on approaches, we have found that the proportion of transcriptionally active RNA polymerase II (active versus total) present throughout the yeast genome is characteristic of some functional gene classes, like those related to ribosomes and mitochondria. This proportion also responds to regulatory stimuli mediated by protein kinase A and, in relation to cytosolic ribosomal-protein genes, it is mediated by the silencing domain of Rap1. We have found that this inactive form of RNA polymerase II, which accumulates along the full length of ribosomal protein genes, is phosphorylated in the Ser5 residue f the CTD, but is hypophosphorylated in Ser2. Using the same experimental approach, we have found that the in vivo-depletion of FACT, a chromatin-related elongation factor, also produces a regulon-specific effect on the expression of the yeast genome. Our work demonstrates that the regulation of transcription elongation is a widespread, gene class-dependent phenomenon which also affects housekeeping genes.
CABD Seminar January 29  @ 12pm
Regulation and degradation of serpin inhibitors which control the immune response in the fly
Dr. David Gubb
Host: Lola Martín-Bermudo
CABD Seminar January 22 @ 12pm
Autophagy in planarians: TOR signalling and beyond
Dr. Cristina González-Estévez
Anne McLaren Fellow Department of Developmental Genetics and Gene Control. Institute of Genetics Queen's Medical Centre University of Nottingham, UK

Summary: The development of a complex multicellular organism requires a careful coordination of growth, cell division, cell differentiation and cell death. All these processes have to happen only in very specific times and places and they have to be perfectly integrated into the whole body plan during development. The case of Tricladida freshwater planarians is very special since they are constantly replacing somatic tissue from a pool of somatic stem cells and are able to regenerate entire functional animals from small starting fragments from almost any part of the body. In addition, they are continuously growing and degrowing their always perfectly scaled bodies according to food availability. Thus, planarians represent an ideal model where to study how these processes are regulated. We are studying the Target of Rapamycin (TOR) signaling pathway known to control cell and organ size in both vertebrates and invertebrates by the integration of various signals that sense environmental conditions, particularly nutrient conditions. Our previous work has already suggested that continued homeostasis and neoblast proliferation requires autophagy, possibly to provide the fuel for continued tissue turnover in the absence of nutrients. Significantly, the conserved autophagy pathway is negatively regulated by TOR signaling. Our results show that planarian TOR complexes are necessary for proper regeneration and adult normal homeostasis.