Exogenous WNT5A and WNT11 proteins rescue CITED2 dysfunction in mouse embryonic stem cells and zebrafish morphants.

Mutations and inadequate methylation profiles of CITED2 are associated with human congenital heart disease (CHD). In mouse, Cited2 is necessary for embryogenesis, particularly for heart development, and its depletion in embryonic stem cells (ESC) impairs cardiac differentiation. We have now determined that Cited2 depletion in ESC affects the expression of transcription factors and cardiopoietic genes involved in early mesoderm and cardiac specification. Interestingly, the supplementation of the secretome prepared from ESC overexpressing CITED2, during the onset of differentiation, rescued the cardiogenic defects of Cited2-depleted ESC. In addition, we demonstrate that the proteins WNT5A and WNT11 held the potential for rescue. We also validated the zebrafish as a model to investigate cited2 function during development. Indeed, the microinjection of morpholinos targeting cited2 transcripts caused developmental defects recapitulating those of mice knockout models, including the increased propensity for cardiac defects and severe death rate. Importantly, the co-injection of anti-cited2 morpholinos with either CITED2 or WNT5A and WNT11 recombinant proteins corrected the developmental defects of Cited2-morphants. This study argues that defects caused by the dysfunction of Cited2 at early stages of development, including heart anomalies, may be remediable by supplementation of exogenous molecules, offering the opportunity to develop novel therapeutic strategies aiming to prevent CHD.

Parallel Genome-wide Profiling of Coding and Non-coding RNAs to Identify Novel Regulatory Elements in Embryonic and Maturated Heart.

Heart development is a complex process, tightly regulated by numerous molecular mechanisms. Key components of the regulatory network underlying heart development are transcription factors (TFs) and microRNAs (miRNAs), yet limited investigation of the role of miRNAs in heart development has taken place. Here, we report the first parallel genome-wide profiling of polyadenylated RNAs and miRNAs in a developing murine heart. These data enable us to identify dynamic activation or repression of numerous biological processes and signaling pathways. More than 200 miRNAs and 25 long non-coding RNAs were differentially expressed during embryonic heart development compared to the mature heart; most of these had not been previously associated with cardiogenesis. Integrative analysis of expression data and potential regulatory interactions suggested 28 miRNAs as novel regulators of embryonic heart development, representing a considerable expansion of the current repertoire of known cardiac miRNAs. To facilitate follow-up investigations, we constructed HeartMiR (http://heartmir.sysbiolab.eu), an open access database and interactive visualization tool for the study of gene regulation by miRNAs during heart development.

FoxN1-dependent thymic epithelial cells promote T-cell leukemia development.

T-cell acute lymphoblastic leukemia (T-ALL) and T-lymphoblastic lymphomas (T-LBL) are aggressive malignancies of thymocytes. The role of thymic microenvironmental cells and stromal factors in thymocyte malignant transformation and T-ALL development remains little explored. Here, using the TEL-JAK2 transgenic (TJ2-Tg) mouse model of T-ALL/LBL, which is driven by constitutive JAK/STAT signaling and characterized by the acquisition of Notch1 mutations, we sought to identify stromal cell alterations associated with thymic leukemogenesis. Immunofluorescence analyses showed that thymic lymphomas presented epithelial areas characterized by keratin (Krt) 5 and Krt8 expression, adjacently to epithelial-free areas negative for Krt expression. Both areas contained abundant laminin (extracellular matrix) and ER-TR7+ (fibroblasts) CD31+ (endothelial) and CD11c+ (dendritic) cells. Besides Krt5, Krt-positive areas harbored medullary thymic epithelial cells (TECs) labeled by Ulex europaeus agglutinin-1. By performing flow cytometry and RNA sequencing analyses of thymic lymphomas, we observed an enrichment in medullary TEC markers in detriment of cortical TEC markers. To assess whether TECs are important for T-ALL/LBL development, we generated TJ2-Tg mice heterozygous for the FoxN1 transcription factor nude null mutation (Foxn1+/nu). Strikingly, in TJ2-Tg;Foxn1+/nu compound mice, both emergence of malignant cells in preleukemic thymi and overt T-ALL onset were significantly delayed. Moreover, in transplantation assays, leukemic cell expansion within the thymus of recipient Foxn1+/nu mice was reduced as compared with control littermates. Since thymopoesis is largely normal in Foxn1+/nu mice, these results indicate that FoxN1 haploinsufficiency in TECs has a more profound impact in thymic leukemogenesis.

StemMapper: a curated gene expression database for stem cell lineage analysis.

Transcriptomic data have become a fundamental resource for stem cell (SC) biologists as well as for a wider research audience studying SC-related processes such as aging, embryonic development and prevalent diseases including cancer, diabetes and neurodegenerative diseases. Access and analysis of the growing amount of freely available transcriptomics datasets for SCs, however, are not trivial tasks. Here, we present StemMapper, a manually curated gene expression database and comprehensive resource for SC research, built on integrated data for different lineages of human and mouse SCs. It is based on careful selection, standardized processing and stringent quality control of relevant transcriptomics datasets to minimize artefacts, and includes currently over 960 transcriptomes covering a broad range of SC types. Each of the integrated datasets was individually inspected and manually curated. StemMapper's user-friendly interface enables fast querying, comparison, and interactive visualization of quality-controlled SC gene expression data in a comprehensive manner. A proof-of-principle analysis discovering novel putative astrocyte/neural SC lineage markers exemplifies the utility of the integrated data resource. We believe that StemMapper can open the way for new insights and advances in SC research by greatly simplifying the access and analysis of SC transcriptomic data. StemMapper is freely accessible at http://stemmapper.sysbiolab.eu.

StemChecker: a web-based tool to discover and explore stemness signatures in gene sets.

Stem cells present unique regenerative abilities, offering great potential for treatment of prevalent pathologies such as diabetes, neurodegenerative and heart diseases. Various research groups dedicated significant effort to identify sets of genes-so-called stemness signatures-considered essential to define stem cells. However, their usage has been hindered by the lack of comprehensive resources and easy-to-use tools. For this we developed StemChecker, a novel stemness analysis tool, based on the curation of nearly fifty published stemness signatures defined by gene expression, RNAi screens, Transcription Factor (TF) binding sites, literature reviews and computational approaches. StemChecker allows researchers to explore the presence of stemness signatures in user-defined gene sets, without carrying-out lengthy literature curation or data processing. To assist in exploring underlying regulatory mechanisms, we collected over 80 target gene sets of TFs associated with pluri- or multipotency. StemChecker presents an intuitive graphical display, as well as detailed statistical results in table format, which helps revealing transcriptionally regulatory programs, indicating the putative involvement of stemness-associated processes in diseases like cancer. Overall, StemChecker substantially expands the available repertoire of online tools, designed to assist the stem cell biology, developmental biology, regenerative medicine and human disease research community. StemChecker is freely accessible at http://stemchecker.sysbiolab.eu.

Targeting molecular networks for drug research.

The study of molecular networks has recently moved into the limelight of biomedical research. While it has certainly provided us with plenty of new insights into cellular mechanisms, the challenge now is how to modify or even restructure these networks. This is especially true for human diseases, which can be regarded as manifestations of distorted states of molecular networks. Of the possible interventions for altering networks, the use of drugs is presently the most feasible. In this mini-review, we present and discuss some exemplary approaches of how analysis of molecular interaction networks can contribute to pharmacology (e.g., by identifying new drug targets or prediction of drug side effects), as well as list pointers to relevant resources and software to guide future research. We also outline recent progress in the use of drugs for in vitro reprogramming of cells, which constitutes an example par excellence for altering molecular interaction networks with drugs.

StemCellNet: an interactive platform for network-oriented investigations in stem cell biology.

Stem cells are characterized by their potential for self-renewal and their capacity to differentiate into mature cells. These two key features emerge through the interplay of various factors within complex molecular networks. To provide researchers with a dedicated tool to investigate these networks, we have developed StemCellNet, a versatile web server for interactive network analysis and visualization. It rapidly generates focused networks based on a large collection of physical and regulatory interactions identified in human and murine stem cells. The StemCellNet web-interface has various easy-to-use tools for selection and prioritization of network components, as well as for integration of expression data provided by the user. As a unique feature, the networks generated can be screened against a compendium of stemness-associated genes. StemCellNet can also indicate novel candidate genes by evaluating their connectivity patterns. Finally, an optional dataset of generic interactions, which provides large coverage of the human and mouse proteome, extends the versatility of StemCellNet to other biomedical research areas in which stem cells play important roles, such as in degenerative diseases or cancer. The StemCellNet web server is freely accessible at http://stemcellnet.sysbiolab.eu.

UniHI 7: an enhanced database for retrieval and interactive analysis of human molecular interaction networks.

Unified Human Interactome (UniHI) (http://www.unihi.org) is a database for retrieval, analysis and visualization of human molecular interaction networks. Its primary aim is to provide a comprehensive and easy-to-use platform for network-based investigations to a wide community of researchers in biology and medicine. Here, we describe a major update (version 7) of the database previously featured in NAR Database Issue. UniHI 7 currently includes almost 350,000 molecular interactions between genes, proteins and drugs, as well as numerous other types of data such as gene expression and functional annotation. Multiple options for interactive filtering and highlighting of proteins can be employed to obtain more reliable and specific network structures. Expression and other genomic data can be uploaded by the user to examine local network structures. Additional built-in tools enable ready identification of known drug targets, as well as of biological processes, phenotypes and pathways enriched with network proteins. A distinctive feature of UniHI 7 is its user-friendly interface designed to be utilized in an intuitive manner, enabling researchers less acquainted with network analysis to perform state-of-the-art network-based investigations.