Regulated transcription controls the diversity, developmental pathways and spatial organization of the hundreds of cell types that make up a mammal. Using single-molecule cDNA sequencing, we mapped transcription start sites (TSSs) and their usage in human and mouse primary cells, cell lines and tissues to produce a comprehensive overview of mammalian gene expression across the human body. We find that few genes are truly ‘housekeeping’, whereas many mammalian promoters are composite entities composed of several closely separated TSSs, with independent cell-type-specific expression profiles. TSSs specific to different cell types evolve at different rates, whereas promoters of broadly expressed genes are the most conserved. Promoter-based expression analysis reveals key transcription factors defining cell states and links them to binding-site motifs. The functions of identified novel transcripts can be predicted by coexpression and sample ontology enrichment analyses. The functional annotation of the mammalian genome 5 (FANTOM5) project provides comprehensive expression profiles and functional annotation of mammalian cell-type-specific transcriptomes with wide applications in biomedical research.
Transforming growth factor-beta (TGFbeta) regulates the activation state of the endothelium via two opposing type I receptor/Smad pathways. Activin receptor-like kinase-1 (ALK1) induces Smad1/5 phosphorylation, leading to an increase in endothelial cell proliferation and migration, while ALK5 promotes Smad2/3 activation and inhibits both processes. Here, we report that ALK5 is important for TGFbeta/ALK1 signaling; endothelial cells lacking ALK5 are deficient in TGFbeta/ALK1-induced responses. More specifically, we show that ALK5 mediates a TGFbeta-dependent recruitment of ALK1 into a TGFbeta receptor complex and that the ALK5 kinase activity is required for optimal ALK1 activation. TGFbeta type II receptor is also required for ALK1 activation by TGFbeta. Interestingly, ALK1 not only induces a biological response opposite to that of ALK5 but also directly antagonizes ALK5/Smad signaling.
Endoglin is a transmembrane accessory receptor for transforming growth factor-beta (TGF-beta) that is predominantly expressed on proliferating endothelial cells in culture and on angiogenic blood vessels in vivo. Endoglin, as well as other TGF-beta signalling components, is essential during angiogenesis. Mutations in endoglin and activin receptor-like kinase 1 (ALK1), an endothelial specific TGF-beta type I receptor, have been linked to the vascular disorder, hereditary haemorrhagic telangiectasia. However, the function of endoglin in TGF-beta/ALK signalling has remained unclear. Here we report that endoglin is required for efficient TGF-beta/ALK1 signalling, which indirectly inhibits TGF-beta/ALK5 signalling. Endothelial cells lacking endoglin do not grow because TGF-beta/ALK1 signalling is reduced and TGF-beta/ALK5 signalling is increased. Surviving cells adapt to this imbalance by downregulating ALK5 expression in order to proliferate. The ability of endoglin to promote ALK1 signalling also explains why ectopic endoglin expression in endothelial cells promotes proliferation and blocks TGF-beta-induced growth arrest by indirectly reducing TGF-beta/ALK5 signalling. Our results indicate a pivotal role for endoglin in the balance of ALK1 and ALK5 signalling to regulate endothelial cell proliferation.
Since human embryonic stem cells (hESCs) were first differentiated to beating cardiomyocytes a decade ago, interest in their potential applications has increased exponentially. This has been further enhanced over recent years by the discovery of methods to induce pluripotency in somatic cells, including those derived from patients with hereditary cardiac diseases. Human pluripotent stem cells have been among the most challenging cell types to grow stably in culture but advances in reagent development now mean that most laboratories can expand both embryonic and induced pluripotent stem cells robustly using commercially available products. However, differentiation protocols have lagged behind and, in many cases, only produce the cell types required with low efficiency. Cardiomyocyte differentiation techniques were also initially inefficient and not readily transferable across cell lines, but there are now a number of more robust protocols available. Here we review the basic biology underlying the differentiation of pluripotent cells to cardiac lineages and describe current state-of-the-art protocols as well as ongoing refinements. This should provide a useful entry for laboratories new to this area to start their research. Ultimately, efficient and reliable differentiation methodologies are essential to generate desired cardiac lineages in order to realize the full promise of human pluripotent stem cells for biomedical research, drug development, and clinical applications.
Autotaxin (ATX), or nucleotide pyrophosphatase-phosphodiesterase 2, is a secreted lysophospholipase D that promotes cell migration, metastasis, and angiogenesis. ATX generates lysophosphatidic acid (LPA), a lipid mitogen and motility factor that acts on several G protein-coupled receptors. Here we report that ATX-deficient mice die at embryonic day 9.5 (E9.5) with profound vascular defects in yolk sac and embryo resembling the G␣ 13 knockout phenotype. Furthermore, at E8.5, ATX-deficient embryos showed allantois malformation, neural tube defects, and asymmetric headfolds. The onset of these abnormalities coincided with increased expression of ATX and LPA receptors in normal embryos. ATX heterozygous mice appear healthy but show half-normal ATX activity and plasma LPA levels. Our results reveal a critical role for ATX in vascular development, indicate that ATX is the major LPA-producing enzyme in vivo, and suggest that the vascular defects in ATX-deficient embryos may be explained by loss of LPA signaling through G␣ 13 .Autotaxin (ATX), also known as ectonucleotide pyrophosphatase-phosphodiesterase 2, belongs to the nucleotide pyrophosphatase (NPP) family of ectoenzymes and exoenzymes, originally defined by their ability to hydrolyze nucleotides in vitro (8,15,44). Full-length ATX is cleaved along the classical export pathway and secreted as a catalytically active glycoprotein (21, 52). ATX was initially isolated as an autocrine motility factor for melanoma cells (45) and later found to promote metastasis and tumor vascularization in nude mice as well as eliciting an angiogenic response in Matrigel assays (31, 32). Hence, ATX may contribute to tumor progression by providing an invasive and/or angiogenic microenvironment for both malignant and stromal cells, a notion supported by growing evidence that ATX expression is upregulated in various invasive and metastatic cancers (4,18,22,28,43,55).The physiological substrate of ATX had remained elusive until it was discovered that ATX is identical to lysophospholipase D (lysoPLD), a secreted enzyme present in plasma and conditioned media that converts lysophosphatidylcholine (LPC) into bioactive lysophosphatidic acid (LPA) (11,47,48). LPA stimulates cell proliferation, migration, and survival by acting on specific G protein-coupled receptors (GPCRs) that are linked to multiple G proteins, including G q/11 , G i/o , and G 12/13 (20,30). LPA promotes wound healing in vivo and has been implicated in tumor progression, inflammation, vascular disease, and neural development (5,23,28,42,51). It has now become clear that LPA production, rather than nucleotide metabolism, accounts for the growth factor-like effects of ATX observed in cell culture. Strikingly, the other NPP family members lack intrinsic lysoPLD activity despite the similarity between their catalytic domain and that of ATX (14), implying that ATX/NPP2 is a unique lysoPLD with no functional redundancy within the NPP family.In addition to converting LPC into LPA, ATX can also hydrolyze sphingosyl-phosphorycholine (...
The potential usefulness of human embryonic stem cells for therapy derives from their ability to form any cell in the body. This potential has been used to justify intensive research despite some ethical concerns. In parallel, scientists have searched for adult stem cells that can be used as an alternative to embryonic cells, and, for the heart at least, these efforts have led to promising results. However, most adult cardiomyocytes are unable to divide and form new cardiomyocytes and would therefore be unable to replace those lost as a result of disease. Basic questions--for example, whether cardiomyocyte replacement or alternatives, such as providing the damaged heart with new blood vessels or growth factors to activate resident stem cells, are the best approach--remain to be fully addressed. Despite this, preclinical studies on cardiomyocyte transplantation in animals and the first clinical trials with adult stem cells have recently been published with mixed results.
Defined growth conditions are essential for many applications of human embryonic stem cells (hESC). Most defined media are presently used in combination with Matrigel, a partially defined extracellular matrix (ECM) extract from mouse sarcoma. Here, we defined ECM requirements of hESC by analyzing integrin expression and ECM production and determined integrin function using blocking antibodies. hESC expressed all major ECM proteins and corresponding integrins. We then systematically replaced Matrigel with defined medium supplements and ECM proteins. Cells attached efficiently to natural human vitronectin, fibronectin, and Matrigel but poorly to laminin ؉ entactin and collagen IV. Integrin-blocking antibodies demonstrated that ␣V5 integrins mediated adhesion to vitronectin, ␣51 mediated adhesion to fibronectin, and ␣61 mediated adhesion to laminin ؉ entactin. Fibronectin in feeder cell-conditioned medium partially supported growth on all natural matrices, but in defined, nonconditioned medium only Matrigel or (natural and recombinant) vitronectin was effective. Recombinant vitronectin was the only defined functional alternative to Matrigel, supporting sustained self-renewal and pluripotency in three independent hESC lines. STEM
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