[Show abstract][Hide abstract] ABSTRACT: Gene ontology (GO) enrichment is commonly used for inferring biological meaning from systems biology experiments. However, determining differential GO and pathway enrichment between DNA-binding experiments or using the GO structure to classify experiments has received little attention.
Herein, we present a bioinformatics tool, CompGO, for identifying Differentially Enriched Gene Ontologies, called DiEGOs, and pathways, through the use of a z-score derivation of log odds ratios, and visualizing these differences at GO and pathway level. Through public experimental data focused on the cardiac transcription factor NKX2-5, we illustrate the problems associated with comparing GO enrichments between experiments using a simple overlap approach.
We have developed an R/Bioconductor package, CompGO, which implements a new statistic normally used in epidemiological studies for performing comparative GO analyses and visualizing comparisons from . BED data containing genomic coordinates as well as gene lists as inputs. We justify the statistic through inclusion of experimental data and compare to the commonly used overlap method. CompGO is freely available as a R/Bioconductor package enabling easy integration into existing pipelines and is available at: http://www.bioconductor.org/packages/release/bioc/html/CompGO.html packages/release/bioc/html/CompGO.html.
[Show abstract][Hide abstract] ABSTRACT: To model cardiac gene regulatory networks in health and disease we used DamID to establish robust target gene sets for the cardiac homeodomain factor NKX2-5 and two congenital heart disease-associated mutants carrying a crippled homeodomain, which normally functions as DNA- and protein-binding interface. Despite compromised direct DNA-binding, NKX2-5 mutants retained partial functionality and bound hundreds of targets, including NKX2-5 wild type targets and unique sets of 'off-targets'. NKX2-5∆HD, which lacks the entire homeodomain, could still dimerise with wild type NKX2-5 and its cofactors, including newly-discovered cofactors of the ETS family, through the conserved tyrosine-rich domain (YRD). NKX2-5∆HD off-targets showed overrepresentation of many binding motifs, including ETS motifs, the majority co-occupied by ETS proteins as determined by DamID. Off-targets of an NKX2-5 YRD mutant were not enriched in ETS targets. Our study reveals off-target binding and transcriptional activity for NKX2-5 mutations driven in part by cofactor interactions, suggesting a novel type of gain-of-function in congenital heart disease.
[Show abstract][Hide abstract] ABSTRACT: Congenital heart disease places a significant burden on the individual, family and community despite significant advances in our understanding of aetiology and treatment. Early research in ischaemic heart disease has paved the way for stem cell technology and bioengineering, which promises to improve both structural and functional aspects of disease. Stem cell therapy has demonstrated significant improvements in cardiac function in adults with ischaemic heart disease. This finding, together with promising case studies in the paediatric setting, demonstrates the potential for this treatment in congenital heart disease. Furthermore, induced pluripotent stems cell technology, provides a unique opportunity to address aetiological, as well as therapeutic, aspects of disease.
Journal of Clinical Medicine 04/2015; 4(4):768-781. DOI:10.3390/jcm4040768
[Show abstract][Hide abstract] ABSTRACT: Animal genomes contain a code for construction of the body plan from a fertilized egg. Understanding how genome information is deciphered to create the complex multilayered regulatory systems that drive organismal development, and which become altered in disease, is one of the greatest challenges in the biological sciences. The development of methods that effectively represent and communicate the complexity inherent in gene regulatory networks remains a major barrier. This review introduces the philosophy of systems biology and discusses recent progress in understanding the development of the heart at a systems biology level.
Cold Spring Harbor Perspectives in Medicine 10/2014; 4(11). DOI:10.1101/cshperspect.a013839 · 9.47 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Mammalian hearts carry a number of primitive stem cell-like populations, although the magnitude of their contribution to tissue homeostasis and repair remains controversial. Recent CRE recombinase-based lineage tracing experiments suggest only a minor contribution to the formation of new cardiomyocytes from such cells, albeit one that might be augmented therapeutically. As the field explores clinical translation of cardiac stem cells, it will be important to understand the biology of these cells in great detail. In this review we document the various reported stem and progenitor cell populations in mammalian hearts and discuss the current state of knowledge on their origins and lineage capabilities.
Stem Cell Research 10/2014; 13(3). DOI:10.1016/j.scr.2014.09.008 · 3.69 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Mammalian embryos develop in a low oxygen environment. The transcription factor hypoxia inducible factor 1a (HIF1α) is a key element in the cellular response to hypoxia. Complete deletion of Hif1α from the mouse conceptus causes extensive placental, vascular and heart defects, resulting in embryonic lethality. However the precise role of Hif1α in each of these organ systems remains unknown. To further investigate, we conditionally-deleted Hif1α from mesoderm, vasculature and heart individually. Surprisingly, deletion from these tissues did not recapitulate the same severe heart phenotype or embryonic lethality. Placental insufficiency, such as occurs in the complete Hif1α null, results in elevated cellular hypoxia in mouse embryos. We hypothesized that subjecting the Hif1α conditional null embryos to increased hypoxic stress might exacerbate the effects of tissue-specific Hif1α deletion. We tested this hypothesis using a model system mimicking placental insufficiency. We found that the majority of embryos lacking Hif1α in the heart died when exposed to non-physiological hypoxia. This was a heart-specific phenomenon, as HIF1α protein accumulated predominantly in the myocardium of hypoxia-stressed embryos. Our study demonstrates the vulnerability of the heart to lowered oxygen levels, and that under such conditions of non-physiological hypoxia the embryo absolutely requires Hif1α to continue normal development. Importantly, these findings extend our understanding of the roles of Hif1α in cardiovascular development.
[Show abstract][Hide abstract] ABSTRACT: Donation after circulatory death (DCD) offers a potential additional source of cardiac allografts. We used a porcine asphyxia model to evaluate viability of DCD hearts subjected to warm ischemic times (WIT) of 20–40 min prior to flushing with Celsior (C) solution. We then assessed potential benefits of supplementing C with erythropoietin, glyceryl trinitrate and zoniporide (Cs), a combination that we have shown previously to activate ischemic postconditioning pathways. Hearts flushed with C/Cs were assessed for functional, biochemical and metabolic recovery on an ex vivo working heart apparatus. Hearts exposed to 20-min WIT showed full recovery of functional and metabolic profiles compared with control hearts (no WIT). Hearts subjected to 30- or 40-min WIT prior to C solution showed partial and no recovery, respectively. Hearts exposed to 30-min WIT and Cs solution displayed complete recovery, while hearts exposed to 40-min WIT and Cs solution demonstrated partial recovery. We conclude that DCD hearts flushed with C solution demonstrate complete recovery up to 20-min WIT after which there is rapid loss of viability. Cs extends the limit of WIT tolerability to 30 min. DCD hearts with ≤30-min WIT may be suitable for transplantation and warrant assessment in a transplant model.
American Journal of Transplantation 07/2014; 14(8). DOI:10.1111/ajt.12782 · 5.68 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Risk for schizophrenia is likely to involve gene × environment interactions. Neuregulin 1 (NRG1) is a schizophrenia risk gene, hence any interaction with environmental adversity, such as maternal infection, may provide further insights into the basis of the disease. This study examined the individual and combined effects of prenatal immune activation with Poly I:C and disruption of the schizophrenia risk gene NRG1 on the expression of behavioural phenotypes related to schizophrenia. NRG1 heterozygous (NRG1 HET) mutant breeding pairs were time-mated. Pregnant dams received a single injection (5 mg/kg i.p.) of Poly I:C or vehicle on gestation day 9 (GD9). Offspring were then cross-fostered to vehicle-treated or PolyI:C-treated dams. Expression of schizophrenia-related behavioural endophenotypes was assessed at adolescence and in adulthood. Combining NRG1 disruption and prenatal environmental insult (Poly I:C) caused developmental stage-specific deficits in social behaviour, spatial working memory and PPI. However, combining Poly I:C and cross-fostering produced a number of behavioural deficits in the open field, social behaviour and PPI. This became more complex by combining NRG1 deletion with both Poly I:C exposure and cross-fostering, which had a robust effect on PPI. These findings suggest that concepts of gene × environment interaction in risk for schizophrenia should be elaborated to multiple interactions that involve individual genes interacting with diverse biological and psychosocial environmental factors over early life, to differentially influence particular domains of psychopathology, sometimes over specific stages of development.
[Show abstract][Hide abstract] ABSTRACT: Cardiac progenitor cells (CPCs) must control their number and fate to sustain the rapid heart growth during development, yet the intrinsic factors and environment governing these processes remain unclear. Here, we show that deletion of the ancient cell-fate regulator Numb (Nb) and its homologue Numblike (Nbl) depletes CPCs in second pharyngeal arches (PA2s) and is associated with an atrophic heart. With histological, flow cytometric and functional analyses, we find that CPCs remain undifferentiated and expansive in the PA2, but differentiate into cardiac cells as they exit the arch. Tracing of Nb- and Nbl-deficient CPCs by lineage-specific mosaicism reveals that the CPCs normally populate in the PA2, but lose their expansion potential in the PA2. These findings demonstrate that Nb and Nbl are intrinsic factors crucial for the renewal of CPCs in the PA2 and that the PA2 serves as a microenvironment for their expansion. DOI: http://dx.doi.org/10.7554/eLife.02164.001
[Show abstract][Hide abstract] ABSTRACT: Cardiac fibroblasts are critical to proper heart function through multiple interactions with the myocardial compartment but appreciation of their contribution has suffered from incomplete characterization and lack of cell-specific markers.
To generate an unbiased comparative gene expression profile of the cardiac fibroblast pool, identify and characterize the role of key genes in cardiac fibroblast function, and determine their contribution to myocardial development and regeneration.
High-throughput cell surface and intracellular profiling of cardiac and tail fibroblasts identified canonical MSC and a surprising number of cardiogenic genes, some expressed at higher levels than in whole heart. Whilst genetically marked fibroblasts contributed heterogeneously to interstitial but not cardiomyocyte compartments in infarcted hearts, fibroblast-restricted depletion of one highly expressed cardiogenic marker, Tbx20, caused marked myocardial dysmorphology and perturbations in scar formation upon myocardial infarction.
The surprising transcriptional identity of cardiac fibroblasts, the adoption of cardiogenic gene programs and direct contribution to cardiac development and repair provokes alternative interpretations for studies on more specialized cardiac progenitors, offering a novel perspective for reinterpreting cardiac regenerative therapies.
Circulation Research 03/2014; 114(9). DOI:10.1161/CIRCRESAHA.114.302530 · 11.02 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Congenital heart defects represent the most common human birth defects and are often life-threatening. Frequently, they are caused by abnormalities of the outflow tract whose formation results from coordinated development of cells from mesodermal and neural crest origin and depends on the activity of many different transcription factors. However, place, time, and mode of action have only been analyzed for a few of them. Here we assess the contribution of the closely related high-mobility-group transcription factors Sox4 and Sox11 to outflow tract development and determine their function. Using cell-type-specific deletion in the mouse, we show that Sox11 is required for proper development in both mesodermal cells and neural crest cells. Deletion in either mesoderm or neural crest, or both, leads to outflow tract defects ranging from double outlet right ventricle to common arterial trunk. Sox4 supports Sox11 in its function, but has additional roles with relevance for outflow tract formation in other cell types. The two Sox proteins are dispensable during early phases of cardiac neural crest development including neural tube emigration, proliferation, and migration through the pharyngeal arches. They become essential after arrival of the neural crest cells in the outflow tract for their proper differentiation and interaction with each other as well as with the environment through regulation of cytoskeletal, cell adhesion, and extracellular matrix molecules. Our results demonstrate that Sox4 and Sox11 have multiple functions in several cell types during outflow tract formation and may thus help to understand the basis of congenital heart defects in humans.
Cellular and Molecular Life Sciences CMLS 12/2013; 71(15). DOI:10.1007/s00018-013-1523-x · 5.81 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The pharyngeal arch arteries (PAAs) are transient embryonic blood vessels that make indispensable contributions to the carotid arteries and great vessels of the heart, including the aorta and pulmonary arteries. During embryogenesis, the PAAs appear in a craniocaudal sequence to connect pre-existing segments of the primitive circulation after de novo vasculogenic assembly from angioblast precursors. Despite the unique spatiotemporal characteristics of PAA development, the embryonic origins of PAA angioblasts and the genetic factors regulating their emergence remain unknown. Here, we identify the embryonic source of PAA endothelium as nkx2.5(+) progenitors in lateral plate mesoderm long considered to adopt cell fates within the heart exclusively. Further, we report that PAA endothelial differentiation relies on Nkx2.5, a canonical cardiac transcription factor not previously implicated in blood vessel formation. Together, these studies reveal the heart field origin of PAA endothelium and attribute a new vasculogenic function to the cardiac transcription factor Nkx2.5 during great vessel precursor development.