Mathematical modelling plant signalling networks

During the last two decades, molecular genetic studies and the completion of the sequencing of the Arabidopsis thaliana genome have increased knowledge of hormonal regulation in plants. These signal transduction pathways act in concert through gene regulatory and signalling networks whose main components have begun to be elucidated. Our understanding of the resulting cellular processes is hindered by the complex, and sometimes counter-intuitive, dynamics of the networks, which may be interconnected through feedback controls and cross-regulation. Mathematical modelling provides a valuable tool to investigate such dynamics and to perform in silico experiments that may not be easily carried out in a laboratory. In this article, we firstly review general methods for modelling gene and signalling networks and their application in plants. We then describe specific models of hormonal perception and cross-talk in plants. This sub-cellular analysis paves the way for more comprehensive mathematical studies of hormonal transport and signalling in a multi-scale setting

Organellar carbon metabolism is co-ordinated with distinct developmental phases of secondary xylem

Subcellular compartmentation of plant biosynthetic pathways in the mitochondria and plastids requires coordinated regulation of nuclear encoded genes, and the role of these genes has been largely ignored by wood researchers. In this study, we constructed a targeted systems genetics coexpression network of xylogenesis in Eucalyptus using plastid and mitochondrial carbon metabolic genes and compared the resulting clusters to the aspen xylem developmental series. The constructed network clusters reveal the organization of transcriptional modules regulating subcellular metabolic functions in plastids and mitochondria. Overlapping genes between the plastid and mitochondrial networks implicate the common transcriptional regulation of carbon metabolism during xylem secondary growth. We show that the central processes of organellar carbon metabolism are distinctly coordinated across the developmental stages of wood formation and are specifically associated with primary growth and secondary cell wall deposition. We also demonstrate that, during xylogenesis, plastid-targeted carbon metabolism is partially regulated by the central clock for carbon allocation towards primary and secondary xylem growth, and we discuss these networks in the context of previously established associations with wood-related complex traits. This study provides a new resolution into the integration and transcriptional regulation of plastid- and mitochondrial-localized carbon metabolism during xylogenesis

Analysis of the dynamic co-expression network of heart regeneration in the zebrafish.

The zebrafish has the capacity to regenerate its heart after severe injury. While the function of a few genes during this process has been studied, we are far from fully understanding how genes interact to coordinate heart regeneration. To enable systematic insights into this phenomenon, we generated and integrated a dynamic co-expression network of heart regeneration in the zebrafish and linked systems-level properties to the underlying molecular events. Across multiple post-injury time points, the network displays topological attributes of biological relevance. We show that regeneration steps are mediated by modules of transcriptionally coordinated genes, and by genes acting as network hubs. We also established direct associations between hubs and validated drivers of heart regeneration with murine and human orthologs. The resulting models and interactive analysis tools are available at http://infused.vital-it.ch. Using a worked example, we demonstrate the usefulness of this unique open resource for hypothesis generation and in silico screening for genes involved in heart regeneration

An Esrrb and nanog cell fate regulatory module controlled by feed forward loop interactions

Cell fate decisions during development are governed by multi-factorial regulatory mechanisms including chromatin remodeling, DNA methylation, binding of transcription factors to specific loci, RNA transcription and protein synthesis. However, the mechanisms by which such regulatory 'dimensions' coordinate cell fate decisions are currently poorly understood. Here we quantified the multi-dimensional molecular changes that occur in mouse embryonic stem cells (mESCs) upon depletion of Estrogen related receptor beta (Esrrb), a key pluripotency regulator. Comparative analyses of expression changes subsequent to depletion of Esrrb or Nanog, indicated that a system of interlocked feed-forward loops involving both factors, plays a central part in regulating the timing of mESC fate decisions. Taken together, our meta-analyses support a hierarchical model in which pluripotency is maintained by an Oct4-Sox2 regulatory module, while the timing of differentiation is regulated by a Nanog-Esrrb module

An integrative view of the regulatory and transcriptional landscapes in mouse hematopoiesis.

Thousands of epigenomic data sets have been generated in the past decade, but it is difficult for researchers to effectively use all the data relevant to their projects. Systematic integrative analysis can help meet this need, and the VISION project was established for validated systematic integration of epigenomic data in hematopoiesis. Here, we systematically integrated extensive data recording epigenetic features and transcriptomes from many sources, including individual laboratories and consortia, to produce a comprehensive view of the regulatory landscape of differentiating hematopoietic cell types in mouse. By using IDEAS as our integrative and discriminative epigenome annotation system, we identified and assigned epigenetic states simultaneously along chromosomes and across cell types, precisely and comprehensively. Combining nuclease accessibility and epigenetic states produced a set of more than 200,000 candidate cis-regulatory elements (cCREs) that efficiently capture enhancers and promoters. The transitions in epigenetic states of these cCREs across cell types provided insights into mechanisms of regulation, including decreases in numbers of active cCREs during differentiation of most lineages, transitions from poised to active or inactive states, and shifts in nuclease accessibility of CTCF-bound elements. Regression modeling of epigenetic states at cCREs and gene expression produced a versatile resource to improve selection of cCREs potentially regulating target genes. These resources are available from our VISION website to aid research in genomics and hematopoiesis.National Institute of Diabetes and Digestive and Kidney Diseases (grant number R24DK106766-01A1), the National Human Genome Research Institute (grant number U54HG006998

Network based meta-analysis prediction of microenvironmental relays involved in stemness of human embryonic stem cells

Background. Human embryonic stem cells (hESCs) are pluripotent cells derived from the inner cell mass of in vitro fertilised blastocysts, which can either be maintained in an undifferentiated state or committed into lineages under determined culture conditions. These cells offer great potential for regenerative medicine, but at present, little is known about the mechanisms that regulate hESC stemness; in particular, the role of cell–cell and cell-extracellular matrix interactions remain relatively unexplored. Methods and Results. In this study we have performed an in silico analysis of cellmicroenvironment interactions to identify novel proteins that may be responsible for the maintenance of hESC stemness. A hESC transcriptome of 8,934 mRNAs was assembled using a meta-analysis approach combining the analysis of microarrays and the use of databases for annotation. The STRING database was utilised to construct a protein–protein interaction network focused on extracellular and transcription factor components contained within the assembled transcriptome. This interactome was structurally studied and filtered to identify a short list of 92 candidate proteins, which may regulate hESC stemness. Conclusion.We hypothesise that this list of proteins, either connecting extracellular components with transcriptional networks, or with hub or bottleneck properties, may contain proteins likely to be involved in determining stemness

Identification of novel key factors of heart development using a systems biology approach

Heart diseases are the leading cause of death worldwide. Although surgical interventions can provide valuable options for treatment, current therapies in cardiovascular medicine only delay disease progression. A main reason for this shortcoming is the limited regenerative capacity of the adult human heart. In contrast to many other tissues and organs, the mammalian heart has very limited regenerative capacity. However, it has been observed that neonatal hearts in mice show remarkable capacity to regenerate lost functional muscle tissue, a capacity that rapidly disappears after the first post-natal week. Therefore, the study of heart development might give crucial cues for cardiac regenerative medicine. Heart development or cardiogenesis is a highly complex process with many components that are finely tuned in a precise manner across time and space. Regulation of gene expression plays an important role in this process. To capture this level of regulation, technologies such as microarrays or next generation sequencing provide powerful tools, as they enable the simultaneous measurement of expression levels of thousands of coding and non-coding genes. Although, it is possible to obtain the expression information of several thousand of genes, there is a clear lack of platforms in which research can scan through this information to develop or generate insightful biological questions in the field of heart study. Hence, this doctoral research work has tried to provide different systems biology approaches in order to offer new insights into gene expression events that occur mainly during heart development. These approaches include: (i) the integration of more than 20 published microarray studies related to cardiogenesis and the development of the HeartEXpress database (http://heartexpress.sysbiolab.eu/) to provide an easy and public access to the integrated data; (ii) the integrative analysis of a genome-wide study profiling coding and noncoding genes during embryonic heart development in vivo; (iii) the assessment of transcription factors and miRNAs previously associated to heart development; (iv) the integration and prioritisation of miRNA-mRNA interactions to identify novel miRNAs, mRNAs or miRNAinteractions with potential impact on cardiogenesis; (v) the development of a web-server called HeartmiR (http://heartmir.sysbiolab.eu/), which enables independent query and visualisation of miRNA-mRNA interactions obtained from the in vivo study; and (vi) comparative analysis of in vivo and in vitro studies to obtain further insights into mRNA, miRNA and miRNA-mRNA interactions during embryonic stem cell differentiation and to clarify how the in vitro experiment can be used as a faithful model to study embryonic heart formation. The main contributions of this research work for the study in the heart field are: 1. The development of HeartEXpress, which is a database that integrates the expression of more than 16400 genes and 130 experimental conditions in both human and mouse; 2. The development of HeartmiR, which is a database profiling the expression of 9211 mRNA and 386 microRNAs during the heart development period (from E10.5 to E19.5) and additionally in adult and old murine heart tissue; 3. Identification of the potential of 165 miRNAs to be involved in heart development using different methods of miRNA candidate prioritisation; 4. Identification of 102 miRNA and 214 putative novel miRNA-mRNA interactions relevant for cardiac cell development in vivo and in vitro. Furthermore, from the top20 miRNA with most interactions, 12 of the miRNA (60%) had been already associated to heart related events, indicating promising results for the remaining 8 miRNAs (40%) In summary, I have developed, implemented and applied different systems biology approaches to analyse both publicly available and new generated experimental data. As result, I was able to identify potential novel coding and non-coding key factors important for cardiogenesis that might be utilised as markers or targets in future cardiac regenerative medicine strategies.As doenças cardíacas são uma das principais causas de morte a nível mundial. Apesar das intervenções cirúrgicas serem uma opção de tratamento viável, as terapias atuais apenas conseguem atrasar a progressão da doença. A principal razão para esta insuficiência é a capacidade regenerativa limitada do coração humano adulto. Em contraste com muitos outros tecidos e órgãos, o coração do mamífero tem uma capacidade de regeneração muito limitada. No entanto, foi observado, em corações de ratinhos neonatais, que ainda existe a incrível capacidade de regenerar tecido cardíaco perdido, a qual desaparece rapidamente após a primeira semana depois do nascimento. Portanto, o estudo do desenvolvimento cardíaco poderá providenciar pistas cruciais para posteriormente aplicar à medicina regenerativa cardíaca. O desenvolvimento cardíaco, também designado por cardiogénese, é um processo altamente complexo que envolve vários intervenientes que são coordenados de uma forma precisa no espaço e no tempo. Para tornar possível a captura deste nível complexo de regulação, tecnologias como os microarrays e next generation sequencing são ferramentas poderosas que permitem a medição simultânea dos níveis de expressão de milhares de genes codificantes e não codificantes. No decorrer deste trabalho, diferentes métodos de biologia de sistemas foram implementados com o objetivo de fornecer um vislumbre da expressão genética que ocorre principalmente durante o desenvolvimento cardíaco. Como tal, foram realizadas: (i) a análise de mais de 20 estudos publicados de microarrays relacionados com o desenvolvimento cardíaco e, consequentemente, desenvolvida a ferramenta online HeartEXpress (http://heartexpress.sysbiolab.eu/) que permite o acesso público à base de dados tratados; (ii) a análise de um estudo do genoma que perfila os genes codificantes e não codificantes durante o desenvolvimento cardíaco embrionário in vivo; (iii) a análise de fatores de transcrição previamente associados com o desenvolvimento cardíaco; (iv) a apreciação de interações miRNA-mRNA para revelar novos miRNAs, mRNAs ou interações miRNA-mRNA que possam potencialmente ter um papel preponderante durante a cardiogénese; (v) o desenvolvimento de uma ferramenta online nomeada HeartmiR (http://heartmir.sysbiolab.eu/), que possibilita a consulta independente das interações obtidas no estudo do desenvolvimento cardíaco embrionário in vivo; e (vi) a análise comparativa de um estudo in vitro com o estudo in vivo para fornecer pistas adicionais sobre os miRNAs, mRNAs e interações miRNA-mRNA durante a diferenciação de células estaminais embrionárias e compreender como o modelo in vitro pode ser utilizado para o estudo do desenvolvimento do coração. As principais contribuições deste trabalho de investigação doutoral na área de estudo do coração são: 1. O desenvolvimento do HeartEXpress, sendo esta uma base de dados que integra a expressão de mais de 16400 genes e 130 condições experimentais tanto em humano como em ratinho; 2. O desenvolvimento do HeartmiR, que é uma base de dados que perfila a expressão de 9211 mRNA e 386 miRNAs durante o desenvolvimento cardíaco (de E10.5 a E19.5) e adicionalmente tecido cardíaco de ratinho em jovem adulto e velho; 3. Identificação de 165 miRNAs que podem estar potencialmente envolvidos no desenvolvimento cardíaco utilizando métodos de prioritização de candidatos; 4. Identificação de 102 miRNAs e 214 potenciais interações miRNA-mRNA relevantes para o desenvolvimento cardíaco celular in vivo e in vitro. Adicionalmente, dos top20 miRNAs com mais interações, 12 deles (60%) já foram associados a eventos cardíacos, indicando que os restantes 8 miRNAs (40%) serão promissores para investigações futuras. Os resultados demonstraram que os diferentes métodos de biologia de sistemas aplicados forneceram resultados interessantes de diferentes formas para estudos adicionais ao desenvolvimento cardíaco e, possivelmente, no futuro aplicar a estudos de medicina regenerativa. Para além dos mRNAs e miRNAs que vão ser apresentados neste trabalho e que já foram posteriormente associados ao desenvolvimento cardíaco de algum modo, muitos outros foram descobertos durante o decorrer deste trabalho com o potencial de serem inovadores na área da regeneração e desenvolvimento cardíaco. Por exemplo, a descoberta de genes co-expressos relacionados com desenvolvimento cardíaco poderão providenciar pistas sobre os padrões de expressão que ocorrem durante os eventos relacionados com o coração. Como é no caso de estudo do Isl1, um conhecido fator de transcrição cardíaco, que apresenta como padrão de co-expressão outros três conhecidos fatores ligados ao desenvolvimento cardíaco tais com o Gata3, Wnt2 e Wnt11. Apresenta ainda a co-expressão com outros genes que potencialmente poderão ser importantes no desenvolvimento cardíaco, nomeadamente três genes Riken (um codificante e dois não-codificantes longos). Em contrapartida, investigar interações miRNA-mRNA também é um passo importante, visto que o desenvolvimento cardíaco é um processo altamente regulado, sendo assim possível indiciar potenciais candidatos miRNAs que possam contribuir para o desenvolvimento cardíaco. Neste estudo foram descobertos 3 miRNAs candidatos no estudo in vivo (Let-7i, mir- 3472 e miR-490-3p) que apresentam um grande número de genes “alvo”, sendo indicados como potenciais reguladores do desenvolvimento cardíaco. Por último, a comparação dos estudos de microarrays in vitro e in vivo também foi um ponto importante, uma vez que forneceu informação fulcral sobre as semelhanças e disparidades entre estas duas distintas experiências laboratoriais, contribuindo para entender se o modelo in vitro será um paradigma apropriado para estudar o desenvolvimento cardíaco. Não obstante, os resultados revelaram ser encorajadores, visto terem sido encontradas interações miRNA-mRNA sobrepostas entre os dois estudos, contendo miRNAs que poderão ser promissores para validações experimentais futuras, como acontece, por exemplo, com o miR-608 que foi encontrado como diferencialmente expresso nos dois estudos – in vitro e in vivo – e tem como alvo seis genes que já foram previamente associados ao desenvolvimento cardíaco. Em suma, este trabalho utilizou diferentes metodologias da biologia de sistemas que permitiram utilizar e analisar informação publicamente disponível e dados laboratoriais que permitiram identificar potenciais fatores-chave codificantes e não-codificantes para a cardiogénese e, possivelmente, aplicar no futuro essa informação em validações laboratoriais e na medicina regenerativa

High resolution temporal transcriptomics of mouse embryoid body development reveals complex expression dynamics of coding and noncoding loci.

Cellular responses to stimuli are rapid and continuous and yet the vast majority of investigations of transcriptional responses during developmental transitions typically use long interval time courses; limiting the available interpretive power. Moreover, such experiments typically focus on protein-coding transcripts, ignoring the important impact of long noncoding RNAs. We therefore evaluated coding and noncoding expression dynamics at unprecedented temporal resolution (6-hourly) in differentiating mouse embryonic stem cells and report new insight into molecular processes and genome organization. We present a highly resolved differentiation cascade that exhibits coding and noncoding transcriptional alterations, transcription factor network interactions and alternative splicing events, little of which can be resolved by long-interval developmental time-courses. We describe novel short lived and cycling patterns of gene expression and dissect temporally ordered gene expression changes in response to transcription factors. We elucidate patterns in gene co-expression across the genome, describe asynchronous transcription at bidirectional promoters and functionally annotate known and novel regulatory lncRNAs. These findings highlight the complex and dynamic molecular events underlying mammalian differentiation that can only be observed though a temporally resolved time course