Hard-wired heterogeneity in blood stem cells revealed using a dynamic regulatory network model.

MOTIVATION: Combinatorial interactions of transcription factors with cis-regulatory elements control the dynamic progression through successive cellular states and thus underpin all metazoan development. The construction of network models of cis-regulatory elements, therefore, has the potential to generate fundamental insights into cellular fate and differentiation. Haematopoiesis has long served as a model system to study mammalian differentiation, yet modelling based on experimentally informed cis-regulatory interactions has so far been restricted to pairs of interacting factors. Here, we have generated a Boolean network model based on detailed cis-regulatory functional data connecting 11 haematopoietic stem/progenitor cell (HSPC) regulator genes. RESULTS: Despite its apparent simplicity, the model exhibits surprisingly complex behaviour that we charted using strongly connected components and shortest-path analysis in its Boolean state space. This analysis of our model predicts that HSPCs display heterogeneous expression patterns and possess many intermediate states that can act as 'stepping stones' for the HSPC to achieve a final differentiated state. Importantly, an external perturbation or 'trigger' is required to exit the stem cell state, with distinct triggers characterizing maturation into the various different lineages. By focusing on intermediate states occurring during erythrocyte differentiation, from our model we predicted a novel negative regulation of Fli1 by Gata1, which we confirmed experimentally thus validating our model. In conclusion, we demonstrate that an advanced mammalian regulatory network model based on experimentally validated cis-regulatory interactions has allowed us to make novel, experimentally testable hypotheses about transcriptional mechanisms that control differentiation of mammalian stem cells. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Desenvolupament d'eines d'alta ressolució per al cribatge (screening) de l'activitat de fàrmacs

Projecte de recerca elaborat a partir d’una estada a la Satandford University, EEUU, entre 2007 i 2009. Els darrers anys, hi ha hagut un avanç espectacular en la tecnologia aplicada a l’anàlisi del genoma i del proteoma (microarrays, PCR quantitativa real time, electroforesis dos dimensions, espectroscòpia de masses, etc.) permetent la resolució de mostres complexes i la detecció quantitativa de diferents gens i proteïnes en un sol experiment. A més a més, la seva importància radica en la capacitat d’identificar potencials dianes terapèutiques i possibles fàrmacs, així com la seva aplicació en el disseny i desenvolupament de noves eines de diagnòstic. L’aplicabilitat de les tècniques actuals, però, està limitada al nivell al que el teixit pot ser disseccionat. Si bé donen valuosa informació sobre expressió de gens i proteïnes implicades en una malaltia o en resposta a un fàrmac per exemple, en cap cas, s’obté una informació in situ ni es pot obtenir informació espacial o una resolució temporal, així com tampoc s’obté informació de sistemes in vivo. L’objectiu d’aquest projecte és desenvolupar i validar un nou microscopi, d’alta resolució, ultrasensible i de fàcil ús, que permeti tant la detecció de metabòlits, gens o proteïnes a la cèl•lula viva en temps real com l’estudi de la seva funció. Obtenint així una descripció detallada de les interaccions entre proteïnes/gens que es donen dins la cèl•lula. Aquest microscopi serà un instrument sensible, selectiu, ràpid, robust, automatitzat i de cost moderat que realitzarà processos de cribatge d’alt rendiment (High throughput screening) genètics, mèdics, químics i farmacèutics (per aplicacions diagnòstiques i de identificació i selecció de compostos actius) de manera més eficient. Per poder realitzar aquest objectius el microscopi farà ús de les més noves tecnologies: 1)la microscopia òptica i d’imatge, per millorar la visualització espaial i la sensibilitat de l’imatge; 2) la utilització de nous mètodes de detecció incloent els més moderns avanços en nanopartícules; 3) la creació de mètodes informàtics per adquirir, emmagatzemar i processar les imatges obtingudes.

Transcriptional regulation of RNA polymerase III in mammalian cells

L'ARN Polymérase III (Pol III) transcrit un ensemble de petits ARN non traduits impliqués dans des processus cellulaires tels que la biosynthèse des protéines, la maturation des ARNs ou le contrôle transcriptionnel. De ce fait, la Pol III joue un rôle important dans la régulation de la croissance et la prolifération cellulaire. L'initiation de la transcription par la Pol III nécessite l'interaction entre des facteurs de transcription et le complexe de la Pol III lui-même. Un sous- complexe de la Pol III, composé de 3 sous-unités, HsRPC3, HsRPC6 et HsRPC7 sert d'intermédiaire dans cette interaction. Dans cette étude, nous avons caractérisé une nouvelle sous-unité de la Pol III, HsRPC7-Like, homologue à HsRPC7. Nous avons montré que ces deux homologues se trouvent spécifiquement chez les vertébrés. Ils proviennent d'un ancêtre commun qui, après duplication il y a 600 millions d'années, a donné naissance à ces deux paralogues. Dans les cellules humaines, deux formes de Pol III coexistent : l'une contientt HsRPC7, l'autre HsRPC7-Like. Nous avons localisé, à l'échelle du génome entier, la présence de ces deux formes de Pol III dans des cellules humaines et dans le foie de souris. Les deux sous-unités ont démontré des caractéristiques identiques, suggérant qu'elles possèdent des fonctions similaires. Cependant, nous avons analysé les motifs d'expression des gènes codant pour RPC7 et RPC7-Like dans des lignées cellulaires dans des conditions variées telles que la concentration de sérum et la densité cellulaire, ainsi que les motifs d'expression dans le foie de souris et des cellules d'hépatocarcinome de souris. Nos résultats suggèrent que l'expression de ces deux sous-untiés varie en fonction de l'activité de prolifération de la cellule. - RNA polymerase III (Pol III) transcribes a set of genes coding for short untranslated RNAs involved in essential cellular processes as for example protein biosynthesis, RNA maturation, and transcriptional control. Thereby Pol III plays an important role in regulating cell growth and proliferation. Initiation of Pol III transcription requires interactions between transcription factors and the Pol III core complex. A Pol III sub-complex composed of three subunits, HsRPC3, HsRPC6, and HsRPC7 mediates this interaction. In this study, we have characterized a new Pol III subunit, HsRPC7-Like, an homologue of HsRPC7. We have shown that these two homologues are specific to vertebrates and originate from an ancestor gene that duplicated 600 mio years ago to give birth to two paralogues. In human cells, two forms of Pol III coexist, one containing HsRPC7 and the other HsRPC7-Like. We have localized, genome-wide, these two Pol III forms in human cells and mouse liver. Both subunits were found on all types of Pol III genes, suggesting that they share similar function. However, we analysed the expression patterns of the RPC7 and RPC7-Like coding genes under various conditions of serum concentration and cell density in different cell lines, as well as expression patterns in mouse liver and mouse hepatocarcinoma cells. Our results suggest that the expression of these two subunits varies with the proliferation rate of the cell.

THE CXCR7/CXCR4/CXCL12 AXIS IN HUMAN NEUROBLASTOMA: INVOLVEMENT IN MALIGNANT PROGRESSION

Le neuroblastome (NB) est la tumeur maligne solide extra-crânienne la plus fréquente chez le jeune enfant. L'évolution clinique est très hétérogène, et les NBs de haut risque échappent encore aux traitements les plus agressifs. Diverses études ont montré que les chimiokines et leurs récepteurs, particulièrement l'axe CXCR4/CXCL12, sont impliqués dans la progression tumorale. Dans le NB, l'expression de CXCR4 est corrélée à un pronostic défavorable. De récentes études ont identifié l'expression d'un autre récepteur, CXCR7, présentant une forte affinité pour le ligand CXCL12. Cependant, son implication potentielle dans l'agressivité des NBs reste encore inconnue. Notre étude a pour objectif d'analyser le rôle de CXCR7 dans le comportement malin du NB, et son influence sur la fonctionnalité de l'axe CXCR4/CXCL12. Les profils d'expression de CXCR7 et CXCL12 ont d'abord été évalués sur un large échantillonnage de tissus de NB, incluant des tissus de tumeurs primaires et de métastases, provenant de 156 patients. CXCL12 est fortement détecté dans les vaisseaux et le stroma des tumeurs. Contrairement à CXCR4, CXCR7 n'est que très faiblement exprimé par les tumeurs indifférenciées. Néanmoins, l'expression de CXCR7 augmente dans les tumeurs matures, et se trouve spécifiquement associée aux cellules neurales différentiées, telles que les cellules ganglionnaires. L'expression de CXCR7 est faiblement détectée dans un nombre réduit de lignées de NB, mais peut-être induite suite à des traitements avec des agents de différenciation in vitro. La surexpression de CXCR7, CXCR4 et une combinaison des deux récepteurs dans les lignées IGR-NB8 et SH-SY5Y a permis l'analyse de leur fonction respective. En réponse à leur ligand commun, chaque récepteur induit l'activation de la voie ERK 1/2, mais pas celle de la voie Akt. Contrairement à CXCR4, l'expression exogène de CXCR7 réduit fortement la prolifération des cellules de NB in vitro, et in vivo dans un modèle d'injection sous-cutanée de. souris immunodéprimées. CXCR7 altère également la migration des cellules induite par l'axe CXCR4/CXCL12. De plus, l'utilisation d'un modèle orthotopique murin a démontré que la croissance tumorale induite par CXCR4 peut être fortement retardée lorsque les deux récepteurs sont co-exprimés dans les cellules de NB. Aucune induction de métastases n'a pu être observée dans ce modèle. Cette étude a permis d'identifier un profil d'expression opposé et des rôles distincts pour CXCR7 et CXCR4 dans le NB. En effet, contrairement à CXCR4, CXCR7 présente des propriétés non tumorigéniques et peut être associé au processus de différenciation du NB. De plus, nos analyses suggèrent que CXCR7 peut réguler les mécanismes induits par CXCR4. Ces données ouvrent donc de nouvelles perspectives de recherche quant au rôle de l'axe CXCR7/CXCR4/CXCL12 dans la biologie des NBs. - Neuroblastoma (NB) is a typical childhood and heterogeneous neoplasm for which efficient targeted therapy for high-risk tumours is not yet identified. The chemokine CXCL12, and its receptors CXCR4 and CXCR7 have been involved in tumour progression and dissemination in various cancer models. In the context of NB, CXCR4 expression is associated to undifferentiated tumours and poor prognosis, while the role of CXCR7, the recently identified second CXCL12 receptor, has not yet been elucidated. In this report, CXCR7 and CXCL12 expression were evaluated using a tissue micro-array (TMA) including 156 primary and 56 metastatic NB tissues. CXCL12 was found to be highly associated to NB vascular and stromal structures. In opposite to the CXCR4 expression pattern, the neural-associated CXCR7 expression was extremely low in undifferentiated tumours, while its expression increased in maturated tissues and was specifically associated to the differentiated neural tumour cells. As determined by RT-PCR, CXCR7 expression was only found in a minority of NB cell lines. Moreover, its expression in two CXCR7-negative NB cell lines was further induce upon treatment with differentiation agents in vitro. The relative roles of the two CXCL12 receptors was further assessed by overexpressing individual CXCR7 or CXCR4 receptors, or a combination of both, in the IGR-NB8 and SH-SY5Y NB cell lines. In vitro functional analyses indicated that, in response to their common ligand, both receptors induced activation of ERK 1/2 cascade, but not Akt signaling pathway. CXCR7 strongly reduced in vitro growth, in contrast to CXCR4. Sub-cutaneous implantations of CXCR7-expressing NB cells showed that CXCR7 also drastically reduced in vivo growth. Moreover, CXCR7 impaired CXCR4-mediated chemotaxis, and altered CXCR4-mediated growth when CXCR4/CXCR7-expressing NB cells were engrafted orthotopically in mouse adrenal gland, a CXCL12-producing environment. In such model, CXCR7 alone, or in association with CXCR4, did not induce NB cell metastatic dissemination. In conclusion, the CXCL12 receptors, CXCR7 and CXCR4, revealed opposite expression patterns and distinct functional roles in NB. While CXCR4 favours NB growth and chemotaxis, CXCR7 elicits anti-tumorigenic properties and may be associated with NB differentiation. Importantly, CXCR7 may act as a negative modulator of CXCR4 signaling, further opening new research perspectives for the role of the global CXCR7/CXCR4/CXCL12 axis in NB.

The Thioredoxin TRX-1 Modulates the Function of the Insulin-Like Neuropeptide DAF-28 during Dauer Formation in Caenorhabditis elegans

Thioredoxins comprise a conserved family of redox regulators involved in many biological processes, including stress resistance and aging. We report that the C. elegans thioredoxin TRX-1 acts in ASJ head sensory neurons as a novel modulator of the insulin-like neuropeptide DAF-28 during dauer formation. We show that increased formation of stress-resistant, long-lived dauer larvae in mutants for the gene encoding the insulin-like neuropeptide DAF-28 requires TRX-1 acting in ASJ neurons, upstream of the insulin-like receptor DAF-2. Genetic rescue experiments demonstrate that redox-independent functions of TRX-1 specifically in ASJ neurons are needed for the dauer formation constitutive (Daf-c) phenotype of daf-28 mutants. GFP reporters of trx-1 and daf-28 show opposing expression patterns in dauers (i.e. trx-1 is up-regulated and daf-28 is down-regulated), an effect that is not observed in growing L2/L3 larvae. In addition, functional TRX-1 is required for the down-regulation of a GFP reporter of daf-28 during dauer formation, a process that is likely subject to DAF-28-mediated feedback regulation. Our findings demonstrate that TRX-1 modulates DAF-28 signaling by contributing to the down-regulation of daf-28 expression during dauer formation. We propose that TRX-1 acts as a fluctuating neuronal signaling modulator within ASJ neurons to monitor the adjustment of neuropeptide expression, including insulin-like proteins, during dauer formation in response to adverse environmental conditions.

Functional characterization of E- and P-cadherin in invasive breast cancer cells

BACKGROUND Alterations in the cadherin-catenin adhesion complexes are involved in tumor initiation, progression and metastasis. However, the functional implication of distinct cadherin types in breast cancer biology is still poorly understood. METHODS To compare the functional role of E-cadherin and P-cadherin in invasive breast cancer, we stably transfected these molecules into the MDA-MB-231 cell line, and investigated their effects on motility, invasion and gene expression regulation. RESULTS Expression of either E- and P-cadherin significantly increased cell aggregation and induced a switch from fibroblastic to epithelial morphology. Although expression of these cadherins did not completely reverse the mesenchymal phenotype of MDA-MB-231 cells, both E- and P-cadherin decreased fibroblast-like migration and invasion through extracellular matrix in a similar way. Moreover, microarray gene expression analysis of MDA-MB-231 cells after expression of E- and P-cadherins revealed that these molecules can activate signaling pathways leading to significant changes in gene expression. Although the expression patterns induced by E- and P-cadherin showed more similarities than differences, 40 genes were differentially modified by the expression of either cadherin type. CONCLUSION E- and P-cadherin have similar functional consequences on the phenotype and invasive behavior of MDA-MB-231 cells. Moreover, we demonstrate for the first time that these cadherins can induce both common and specific gene expression programs on invasive breast cancer cells. Importantly, these identified genes are potential targets for future studies on the functional consequences of altered cadherin expression in human breast cancer.

Analysis of the NTPDase and ecto-5'-nucleotidase profiles in serum-limited Trichomonas vaginalis

Trichomonas vaginalis is a parasite of the human urogenital tract that causes trichomonosis, the most prevalent non-viral sexually transmitted disease. Ectonucleoside triphosphate diphosphohydrolase (NTPDase) family members, which hydrolyse extracellular ATP and ADP and ecto-5′-nucleotidase, which hydrolyses AMP, have been characterised in T. vaginalis. For trichomonad culture, the growth medium is supplemented with 10% serum, which is an important source of nutrients, such as adenosine. Here, we investigated the ATP metabolism of T. vaginalis trophozoites from long-term cultures and clinical isolates under limited bovine serum conditions (1% serum). The specific enzymatic activities were expressed as nmol inorganic phosphate (Pi) released/min/mg protein, the gene expression patterns were determined by reverse transcriptase-polymerase chain reaction, the extracellular adenine nucleotide hydrolysis was analysed by high performance liquid chromatography and the cell cycle analysis was assessed by flow cytometry. Serum limitation led to the profound activation of NTPDase and ecto-5'-nucleotidase activities. Furthermore, the levels of NTPDase A and B transcripts increased and extracellular ATP metabolism was activated, which led to enhanced ATP hydrolysis and the formation of ADP and AMP. Moreover, the cell cycle was arrested at the G0/G1 stage, which suggested adenosine uptake. Our data suggest that under conditions of serum limitation, NTPDase and ecto-5'-nucleotidase play a role in providing the adenosine required for T. vaginalis growth and that this process contributes to the establishment of parasitism.

Fine mapping and functional analysis of the multiple sclerosis risk gene CD6.

CD6 has recently been identified and validated as risk gene for multiple sclerosis (MS), based on the association of a single nucleotide polymorphism (SNP), rs17824933, located in intron 1. CD6 is a cell surface scavenger receptor involved in T-cell activation and proliferation, as well as in thymocyte differentiation. In this study, we performed a haptag SNP screen of the CD6 gene locus using a total of thirteen tagging SNPs, of which three were non-synonymous SNPs, and replicated the recently reported GWAS SNP rs650258 in a Spanish-Basque collection of 814 controls and 823 cases. Validation of the six most strongly associated SNPs was performed in an independent collection of 2265 MS patients and 2600 healthy controls. We identified association of haplotypes composed of two non-synonymous SNPs [rs11230563 (R225W) and rs2074225 (A257V)] in the 2(nd) SRCR domain with susceptibility to MS (P max(T) permutation = 1×10(-4)). The effect of these haplotypes on CD6 surface expression and cytokine secretion was also tested. The analysis showed significantly different CD6 expression patterns in the distinct cell subsets, i.e. - CD4(+) naïve cells, P = 0.0001; CD8(+) naïve cells, P<0.0001; CD4(+) and CD8(+) central memory cells, P = 0.01 and 0.05, respectively; and natural killer T (NKT) cells, P = 0.02; with the protective haplotype (RA) showing higher expression of CD6. However, no significant changes were observed in natural killer (NK) cells, effector memory and terminally differentiated effector memory T cells. Our findings reveal that this new MS-associated CD6 risk haplotype significantly modifies expression of CD6 on CD4(+) and CD8(+) T cells.

Emergence of young human genes after a burst of retroposition in primates.

The origin of new genes through gene duplication is fundamental to the evolution of lineage- or species-specific phenotypic traits. In this report, we estimate the number of functional retrogenes on the lineage leading to humans generated by the high rate of retroposition (retroduplication) in primates. Extensive comparative sequencing and expression studies coupled with evolutionary analyses and simulations suggest that a significant proportion of recent retrocopies represent bona fide human genes. We estimate that at least one new retrogene per million years emerged on the human lineage during the past approximately 63 million years of primate evolution. Detailed analysis of a subset of the data shows that the majority of retrogenes are specifically expressed in testis, whereas their parental genes show broad expression patterns. Consistently, most retrogenes evolved functional roles in spermatogenesis. Proteins encoded by X chromosome-derived retrogenes were strongly preserved by purifying selection following the duplication event, supporting the view that they may act as functional autosomal substitutes during X-inactivation of late spermatogenesis genes. Also, some retrogenes acquired a new or more adapted function driven by positive selection. We conclude that retroduplication significantly contributed to the formation of recent human genes and that most new retrogenes were progressively recruited during primate evolution by natural and/or sexual selection to enhance male germline function.

Genome-Wide Analysis of Salicylate and Dibenzofuran Metabolism in Sphingomonas Wittichii RW1.

Sphingomonas wittichii RW1 is a bacterium isolated for its ability to degrade the xenobiotic compounds dibenzodioxin and dibenzofuran (DBF). A number of genes involved in DBF degradation have been previously characterized, such as the dxn cluster, dbfB, and the electron transfer components fdx1, fdx3, and redA2. Here we use a combination of whole genome transcriptome analysis and transposon library screening to characterize RW1 catabolic and other genes implicated in the reaction to or degradation of DBF. To detect differentially expressed genes upon exposure to DBF, we applied three different growth exposure experiments, using either short DBF exposures to actively growing cells or growing them with DBF as sole carbon and energy source. Genome-wide gene expression was examined using a custom-made microarray. In addition, proportional abundance determination of transposon insertions in RW1 libraries grown on salicylate or DBF by ultra-high throughput sequencing was used to infer genes whose interruption caused a fitness loss for growth on DBF. Expression patterns showed that batch and chemostat growth conditions, and short or long exposure of cells to DBF produced very different responses. Numerous other uncharacterized catabolic gene clusters putatively involved in aromatic compound metabolism increased expression in response to DBF. In addition, only very few transposon insertions completely abolished growth on DBF. Some of those (e.g., in dxnA1) were expected, whereas others (in a gene cluster for phenylacetate degradation) were not. Both transcriptomic data and transposon screening suggest operation of multiple redundant and parallel aromatic pathways, depending on DBF exposure. In addition, increased expression of other non-catabolic genes suggests that during initial exposure, S. wittichii RW1 perceives DBF as a stressor, whereas after longer exposure, the compound is recognized as a carbon source and metabolized using several pathways in parallel.

Connexins in lymphatic vessel physiology and disease.

Connexins are transmembrane proteins that form gap junction- and hemi-channels. Once inserted into the membrane, hemi-channels (connexons) allow for diffusion of ions and small molecules (<1kDa) between the extracellular space and the cytosol. Gap junction channels allow diffusion of similar molecules between the cytoplasms of adjacent cells. The expression and function of connexins in blood vessels has been intensely studied in the last few decades. In contrast, only a few studies paid attention to lymphatic vessels; convincing in vivo data with respect to expression patterns of lymphatic connexins and their functional roles have only recently begun to emerge. Interestingly, mutations in connexin genes have been linked to diseases of lymphatic vasculature, most notably primary and secondary lymphedema. This review summarizes the available data regarding lymphatic connexins. More specifically it addresses (i) early studies aimed at presence of gap junction-like structures in lymphatic vessels, (ii) more recent studies focusing on lymphatic connexins using genetically engineered mice, and (iii) results of clinical studies that have reported lymphedema-linked mutations in connexin genes.

The biochemistry of drug metabolism-an introduction: part 7. Intra-individual factors affecting drug metabolism.

This review on intra-individual factors affecting drug metabolism completes our series on the biochemistry of drug metabolism. The article presents the molecular mechanisms causing intra-individual differences in enzyme expression and activity. They include enzyme induction by transcriptional activation and enzyme inhibition on the protein level. The influencing factors are of physiological, pathological, or external origin. Tissue characteristics and developmental age strongly influence enzyme-expression patterns. Further influencing factors are pregnancy, disease, or biological rhythms. Xenobiotics, drugs, constituents of herbal remedies, food constituents, ethanol, and tobacco can all influence enzyme expression or activity and, hence, affect drug metabolism.

Functional diversification of duplicate genes through subcellular adaptation of encoded proteins.

BACKGROUND: Gene duplication is the primary source of new genes with novel or altered functions. It is known that duplicates may obtain these new functional roles by evolving divergent expression patterns and/or protein functions after the duplication event. Here, using yeast (Saccharomyces cerevisiae) as a model organism, we investigate a previously little considered mode for the functional diversification of duplicate genes: subcellular adaptation of encoded proteins. RESULTS: We show that for 24-37% of duplicate gene pairs derived from the S. cerevisiae whole-genome duplication event, the two members of the pair encode proteins that localize to distinct subcellular compartments. The propensity of yeast duplicate genes to evolve new localization patterns depends to a large extent on the biological function of their progenitor genes. Proteins involved in processes with a wider subcellular distribution (for example, catabolism) frequently evolved new protein localization patterns after duplication, whereas duplicate proteins limited to a smaller number of organelles (for example, highly expressed biosynthesis/housekeeping proteins with a slow rate of evolution) rarely relocate within the cell. Paralogous proteins evolved divergent localization patterns by partitioning of ancestral localizations ('sublocalization'), but probably more frequently by relocalization to new compartments ('neolocalization'). We show that such subcellular reprogramming may occur through selectively driven substitutions in protein targeting sequences. Notably, our data also reveal that relocated proteins functionally adapted to their new subcellular environments and evolved new functional roles through changes of their physico-chemical properties, expression levels, and interaction partners. CONCLUSION: We conclude that protein subcellular adaptation represents a common mechanism for the functional diversification of duplicate genes.

vHOG, a multispecies vertebrate ontology of homologous organs groups.

MOTIVATION: Most anatomical ontologies are species-specific, whereas a framework for comparative studies is needed. We describe the vertebrate Homologous Organs Groups ontology, vHOG, used to compare expression patterns between species.¦RESULTS: vHOG is a multispecies anatomical ontology for the vertebrate lineage. It is based on the HOGs used in the Bgee database of gene expression evolution. vHOG version 1.4 includes 1184 terms, follows OBO principles and is based on the Common Anatomy Reference Ontology (CARO). vHOG only describes structures with historical homology relations between model vertebrate species. The mapping to species-specific anatomical ontologies is provided as a separate file, so that no homology hypothesis is stated within the ontology itself. Each mapping has been manually reviewed, and we provide support codes and references when available. Availability and implementation: vHOG is available from the Bgee download site (http://bgee.unil.ch/), as well as from the OBO Foundry and the NCBO Bioportal websites.¦CONTACT: bgee@isb-sib.ch; frederic.bastian@unil.ch.

New gene functions in megakaryopoiesis and platelet formation.

Platelets are the second most abundant cell type in blood and are essential for maintaining haemostasis. Their count and volume are tightly controlled within narrow physiological ranges, but there is only limited understanding of the molecular processes controlling both traits. Here we carried out a high-powered meta-analysis of genome-wide association studies (GWAS) in up to 66,867 individuals of European ancestry, followed by extensive biological and functional assessment. We identified 68 genomic loci reliably associated with platelet count and volume mapping to established and putative novel regulators of megakaryopoiesis and platelet formation. These genes show megakaryocyte-specific gene expression patterns and extensive network connectivity. Using gene silencing in Danio rerio and Drosophila melanogaster, we identified 11 of the genes as novel regulators of blood cell formation. Taken together, our findings advance understanding of novel gene functions controlling fate-determining events during megakaryopoiesis and platelet formation, providing a new example of successful translation of GWAS to function.