Transcriptome analysis identifies stem cells and immune related genes in the cnidarian Hydractinia echinata

An increasing amount of Expressed Sequence Tag (EST) and genomic data predominantly for the cnidarians Acropora, Hydra and Nematostella, reveals that despite being one of the morphologically simplest multicellular animals, cnidarians possess a high genomic complexity. In order to contribute towards a broader coverage of this phylum, an EST project was performed to analyze the transcriptome of Hydractinia echinata. Moreover, transcriptional profiling experiments were carried out to characterize the i-cell population and the immune system of the hydroid. In this work a cDNA-library containing about 20,000 clones was constructed, which covers the entire life cycle of the organism and also represents some stress-induced conditions. After randomly sequencing almost 9,000 clones, EST characterization revealed a broad diversity of genes, with higher sequence similarity to vertebrates than to ecdysozoan invertebrates. Furthermore, a significant number of sequences hitherto unknown in metazoans were detected. The identification of unique Hydractinia sequences is consistent with the suggested high diversity and complexity of genes within the phylum. To store all the acquired information a database aimed at making the data widely available was created, which is accessible at www.mchips.org/hydractinia_echinata.html. To further characterize Hydractinia genes, a cDNA-microarray was constructed including the already sequenced ESTs as well as PCR-products from almost 5,000 un-sequenced cDNAs. Genes associated with the i-cell lineage were identified by the analysis of the gene expression profile of colonies depleted from their i-cells using mitomycin-C and colonies after the recovery from the treatment. Microarray normalized data ended up with 162 significant differentially expressed genes. Several growth and transcription factors as well as genes associated with undifferentiated cells were identified including; BMPs, Bzip/Mafl and CnPL10. In addition, i-cell depleted organisms exhibited an activation of genes involved in detoxification and wound healing activities. These genes are good candidates to define the i-cell population of Hydractinia. Genes associated with the immune system of Hydractinia were identified by the analysis of the expression profile of organisms having a LPS mimicked Gram-negative bacterial infection as well as an allogeneic reaction. 245 candidate genes with a significantly different expression level were identified. Genes associated with an LPS response encode for e.g. HSP70, lipocalin-like proteins, serine protease inhibitors, proteins with TSR domains and lectins. In the case of allorecognition, a probable whole genome response with up-and down regulation of hundreds of genes was observed; demonstrating a complex process. Some of the identified genes encode for e.g. minicollagens, transcriptional and growth factors, proteins with a protective function against oxygen metabolites or with potent inflammatory and neurotoxicity effects. Gene expression pattern analysis provided insights towards the function of many genes which are still unknown. In the case of genes with a known functional annotation, the microarray experiments either corroborated their characterization or defined an alternative one for Hydractinia. This project is the first high-throughput effort aimed to identify and characterize the transcriptome of the colonial marine hydroid Hydractinia echinata. The combination of the EST dataset, database and the microarray, provides a solid platform to promote and facilitate molecular research not only in Hydractinia but also in other cnidarians

Embryonic stem cell-derived hemangioblasts remain epigenetically plastic and require PRC1 to prevent neural gene expression.

Many lineage-specific developmental regulator genes are transcriptionally primed in embryonic stem (ES) cells; RNA Pol(II) is bound at their promoters but is prevented from productive elongation by the activity of polycomb repressive complexes (PRC) 1 and 2. This epigenetically poised state is thought to enable ES cells to rapidly execute multiple differentiation programs and is recognized by a simultaneous enrichment for trimethylation of lysine 4 and trimethylation of lysine 27 of histone H3 (bivalent chromatin) across promoter regions. Here we show that the chromatin profile of this important cohort of genes is progressively modified as ES cells differentiate toward blood-forming precursors. Surprisingly however, neural specifying genes, such as Nkx2-2, Nkx2-9, and Sox1, remain bivalent and primed even in committed hemangioblasts, as conditional deletion of PRC1 results in overt and inappropriate expression of neural genes in hemangioblasts. These data reinforce the importance of PRC1 for normal hematopoietic differentiation and reveal an unexpected epigenetic plasticity of mesoderm-committed hemangioblasts

Desarrollo de una elisa para la detección de anticuerpos IgM anti.ipnv en salmón

Tesis (Ingeniero en Acuicultura)Las pérdidas económicas generadas por el virus de la necrosis pancreática infecciosa (IPNV) han conllevado al desarrollo de técnicas para la rápida detección del patógeno y al control de la enfermedad mediante el uso de vacunas. En este contexto, se estudió el desarrollo de un ELISA que permitiría evaluar la respuesta inmune de los peces inmunizados. El primer objetivo fue caracterizar un panel de anticuerpos monoclonales anti-lgM de salmónidos, del cual se seleccionó el anticuerpo 3H7/E1 . Este anticuerpo fue exitosamente conjugado con peroxidasa , constituyendo uno de los componentes importantes en el desarrollo del ELISA. Además se determinó la concentración óptima de virus necesario para la activación de la placa, y la dilución del anticuerpo monoclonal anti-lgM conjugado a peroxidasa. Para la versión indirecta del ELISA, también se evaluó la dilución adecuada de un anti-lgG de ratón conjugado a peroxidasa. Para evaluar el ensayo se utilizó un panel de 49 sueros provenientes de peces vacunados con una virina del virus, y de 50 sueros de peces controles no vacunados. Al asumir un valor de corte ("cut-off') para el ensayo de ELISA de 0,3 0 .0 . a 450 nm, el grupo inmunizado presento un 89% de lecturas por sobre dicho valor, lo que sería equivalente a peces positivos. En el grupo control sólo un 44% de los sueros presentaba títulos de lgM altos contra el virus. Sin embargo, sueros que poseían altas lecturas de anticuerpos antiIPNV reaccionaron positivamente en placas activadas con Piscirickettsia sa/monis. Para determinar la especificidad de las lgM, estas fueron absorbidas con el virus, P. salmonis y otros patógenos de peces. Al absorber las lgM con los otros patógenos de peces se encontró que los títulos contra el virus o contra P. salmonis no fueron afectados. Sin embargo, los resultados de absorción con el virus o con P. salmonis indicaron que existe reacción cruzada de los sueros entre ambos patógenos, lo que sugiere que las lgM presentes en el suero son inespecíficas o reaccionan con epítopos de algunos antígenos que son comunes al virus y a P. salmonis.The economic loss generated by the infectious necrosis pancreatic virus (IPNV) has lead to the development of techniques for fast detection of the pathogen and to control the disease by vaccination. In this context, the development of an ELISA which may allow us to evaluate the immune response of immunised fish has been studied. The first objective was to characterize a panel of monoclonal anti-salmon lgM antibodies, from which the 3H7/E1 antibody was selected. This antibody was successfully conjugated with peroxidasa, being one of the important component for the development of the ELISA. Furthermore, the optimal concentration of virus needed to activate the plate and the optimal dilution of the monoclonal anti-lgM antibody conjugated with peroxidasa was determinated. For the indirect version of the ELISA, we also evaluated the optimal dilution of a mouse anti-lgG antibody conjugated with peroxidasa. To evaluate the assay we used a panel of 49 fish sera that were vaccinated whit a virine vaccine against IPNV, and 50 sera of non immunized control fish. A "cut-off' value of 0,3 0.0. at 450 nm was assumed. The vaccinated group showed a 89% of lectures over that value, considering these fish as positives. In the control group, only 44 % of the serum presented high titres against the virus. However, serum that have positives lectures of antiIPNV antibodies reacted positively in ELISA plate activated with Piscirickettsia sa/monis. To determinate the lgM specifity, these sera were absorbed with the virus, P. sa/monis and other fish pathogens. When the serum was absorbed with the other fish pathogens, we found that the titres against the virus or against P. salmonis were not affected . Nevertheless, the results of the absorption with the virus or with P. salmonis showed that cross-reaction of the serum between these two pathogens exists, and suggesting that the lgM present in the serum are either non specific or reacted with sorne epitopes of antigen common to the virus and P. salmonis

The transcriptome of the colonial marine hydroid hydractinia echinata

An increasing amount of expressed sequence tag (EST) and genomic data, predominantly for the cnidarians Acropora, Hydra and Nematostella, reveals that cnidarians have a high genomic complexity, despite being one of the morphologically simplest multicellular animals. Considering the diversity of cnidarians, we performed an EST project on the hydroid Hydractinia echinata, to contribute towards a broader coverage of this phylum. After random sequencing of almost 9000 clones, EST characterization revealed a broad diversity in gene content. Corroborating observations in other cnidarians, Hydractinia sequences exhibited a higher sequence similarity to vertebrates than to ecdysozoan invertebrates. A significant number of sequences were hitherto undescribed in metazoans, suggesting that these may be either cnidarian innovations or ancient genes lost in the bilaterian genomes analysed so far. However, we cannot rule out some degree of contamination from commensal bacteria. The identification of unique Hydractinia sequences emphasizes that the acquired genomic information generated so far is not large enough to be representative of the highly diverse cnidarian phylum. Finally, a database was created to store all the acquired information (http://www.mchips.org/hydractinia_echinata.html)