Transcription factors regulating vasculogenesis and angiogenesis.

Transcription factors (TFs) play a crucial role in regulating the dynamic and precise patterns of gene expression required for the initial specification of endothelial cells (ECs), and during endothelial growth and differentiation. While sharing many core features, ECs can be highly heterogeneous. Differential gene expression between ECs is essential to pattern the hierarchical vascular network into arteries, veins and capillaries, to drive angiogenic growth of new vessels, and to direct specialization in response to local signals. Unlike many other cell types, ECs have no single master regulator, instead relying on differing combinations of a necessarily limited repertoire of TFs to achieve tight spatial and temporal activation and repression of gene expression. Here, we will discuss the cohort of TFs known to be involved in directing gene expression during different stages of mammalian vasculogenesis and angiogenesis, with a primary focus on development.

Finding and Verifying Enhancers for Endothelial-Expressed Genes.

Identification and analysis of enhancers for endothelial-expressed genes can provide crucial information regarding their upstream transcriptional regulators. However, enhancer identification can be challenging, particularly for people with limited access or experience of bioinformatics, and transgenic analysis of enhancer activity patterns can be prohibitively expensive. Here we describe how to use publicly available datasets displayed on the UCSC Genome Browser to identify putative endothelial enhancers for mammalian genes. Furthermore, we detail how to utilize mosaic Tol2-mediated transgenesis in zebrafish to verify whether a putative enhancer is capable of directing endothelial-specific patterns of gene expression.

ETS factors are required but not sufficient for specific patterns of enhancer activity in different endothelial subtypes.

Correct vascular differentiation requires distinct patterns of gene expression in different subtypes of endothelial cells. Members of the ETS transcription factor family are essential for the transcriptional activation of arterial and angiogenesis-specific gene regulatory elements, leading to the hypothesis that they play lineage-defining roles in arterial and angiogenic differentiation directly downstream of VEGFA signalling. However, an alternative explanation is that ETS binding at enhancers and promoters is a general requirement for activation of many endothelial genes regardless of expression pattern, with subtype-specificity provided by additional factors. Here we use analysis of Ephb4 and Coup-TFII (Nr2f2) vein-specific enhancers to demonstrate that ETS factors are equally essential for vein, arterial and angiogenic-specific enhancer activity patterns. Further, we show that ETS factor binding at these vein-specific enhancers is enriched by VEGFA signalling, similar to that seen at arterial and angiogenic enhancers. However, while arterial and angiogenic enhancers can be activated by VEGFA in vivo, the Ephb4 and Coup-TFII venous enhancers are not, suggesting that the specificity of VEGFA-induced arterial and angiogenic enhancer activity occurs via non-ETS transcription factors. These results support a model in which ETS factors are not the primary regulators of specific patterns of gene expression in different endothelial subtypes.

Regulatory pathways governing murine coronary vessel formation are dysregulated in the injured adult heart.

The survival of ischaemic cardiomyocytes after myocardial infarction (MI) depends on the formation of new blood vessels. However, endogenous neovascularization is inefficient and the regulatory pathways directing coronary vessel growth are not well understood. Here we describe three independent regulatory pathways active in coronary vessels during development through analysis of the expression patterns of differentially regulated endothelial enhancers in the heart. The angiogenic VEGFA-MEF2 regulatory pathway is predominantly active in endocardial-derived vessels, whilst SOXF/RBPJ and BMP-SMAD pathways are seen in sinus venosus-derived arterial and venous coronaries, respectively. Although all developmental pathways contribute to post-MI vessel growth in the neonate, none are active during neovascularization after MI in adult hearts. This was particularly notable for the angiogenic VEGFA-MEF2 pathway, otherwise active in adult hearts and during neoangiogenesis in other adult settings. Our results therefore demonstrate a fundamental divergence between the regulation of coronary vessel growth in healthy and ischemic adult hearts.

Venous identity requires BMP signalling through ALK3.

Venous endothelial cells are molecularly and functionally distinct from their arterial counterparts. Although veins are often considered the default endothelial state, genetic manipulations can modulate both acquisition and loss of venous fate, suggesting that venous identity is the result of active transcriptional regulation. However, little is known about this process. Here we show that BMP signalling controls venous identity via the ALK3/BMPR1A receptor and SMAD1/SMAD5. Perturbations to TGF-ß and BMP signalling in mice and zebrafish result in aberrant vein formation and loss of expression of the venous-specific gene Ephb4, with no effect on arterial identity. Analysis of a venous endothelium-specific enhancer for Ephb4 shows enriched binding of SMAD1/5 and a requirement for SMAD binding motifs. Further, our results demonstrate that BMP/SMAD-mediated Ephb4 expression requires the venous-enriched BMP type I receptor ALK3/BMPR1A. Together, our analysis demonstrates a requirement for BMP signalling in the establishment of Ephb4 expression and the venous vasculature.

Endothelial-Specific Cre Mouse Models.

The field of vascular biology has gained enormous insight from the use of Cre and inducible Cre mouse models to temporally and spatially manipulate gene expression within the endothelium. Models are available to constitutively or inducibly modulate gene expression in all or a specified subset of endothelial cells. However, caution should be applied to both the selection of allele and the analysis of resultant phenotype: many similarly named Cre models have divergent activity patterns while ectopic or inconsistent Cre or inducible Cre expression can dramatically affect results. In an effort to disambiguate previous data and to provide a resource to aid appropriate experimental design, here we summarize what is known about Cre recombinase activity in the most widely used endothelial-specific Cre and Cre/ERT2 mouse models.

Differential tissue specific, temporal and spatial expression patterns of the Aggrecan gene is modulated by independent enhancer elements.

The transcriptional mechanism through which chondrocytes control the spatial and temporal composition of the cartilage tissue has remained largely elusive. The central aim of this study was to identify whether transcriptional enhancers played a role in the organisation of the chondrocytes in cartilaginous tissue. We focused on the Aggrecan gene (Acan) as it is essential for the normal structure and function of cartilage and it is expressed developmentally in different stages of chondrocyte maturation. Using transgenic reporter studies in mice we identified four elements, two of which showed individual chondrocyte developmental stage specificity. In particular, one enhancer (-80) distinguishes itself from the others by being predominantly active in adult cartilage. Furthermore, the -62 element uniquely drove reporter activity in early chondrocytes. The remaining chondrocyte specific enhancers, +28 and -30, showed no preference to chondrocyte type. The transcription factor SOX9 interacted with all the enhancers in vitro and mutation of SOX9 binding sites in one of the enhancers (-30) resulted in a loss of its chondrocyte specificity and ectopic enhancer reporter activity. Thus, the Acan enhancers orchestrate the precise spatiotemporal expression of this gene in cartilage types at different stages of development and adulthood.

SoxF factors induce Notch1 expression via direct transcriptional regulation during early arterial development.

Arterial specification and differentiation are influenced by a number of regulatory pathways. While it is known that the Vegfa-Notch cascade plays a central role, the transcriptional hierarchy controlling arterial specification has not been fully delineated. To elucidate the direct transcriptional regulators of Notch receptor expression in arterial endothelial cells, we used histone signatures, DNaseI hypersensitivity and ChIP-seq data to identify enhancers for the human NOTCH1 and zebrafish notch1b genes. These enhancers were able to direct arterial endothelial cell-restricted expression in transgenic models. Genetic disruption of SoxF binding sites established a clear requirement for members of this group of transcription factors (SOX7, SOX17 and SOX18) to drive the activity of these enhancers in vivo Endogenous deletion of the notch1b enhancer led to a significant loss of arterial connections to the dorsal aorta in Notch pathway-deficient zebrafish. Loss of SoxF function revealed that these factors are necessary for NOTCH1 and notch1b enhancer activity and for correct endogenous transcription of these genes. These findings position SoxF transcription factors directly upstream of Notch receptor expression during the acquisition of arterial identity in vertebrates.

An Intronic Flk1 Enhancer Directs Arterial-Specific Expression via RBPJ-Mediated Venous Repression.

OBJECTIVE: The vascular endothelial growth factor (VEGF) receptor Flk1 is essential for vascular development, but the signaling and transcriptional pathways by which its expression is regulated in endothelial cells remain unclear. Although previous studies have identified 2 Flk1 regulatory enhancers, these are dispensable for Flk1 expression, indicating that additional enhancers contribute to Flk1 regulation in endothelial cells. In the present study, we sought to identify Flk1 enhancers contributing to expression in endothelial cells. APPROACH AND RESULTS: A region of the 10th intron of the Flk1 gene (Flk1in10) was identified as a putative enhancer and tested in mouse and zebrafish transgenic models. This region robustly directed reporter gene expression in arterial endothelial cells. Using a combination of targeted mutagenesis of transcription factor-binding sites and gene silencing of transcription factors, we found that Gata and Ets factors are required for Flk1in10 enhancer activity in all endothelial cells. Furthermore, we showed that activity of the Flk1in10 enhancer is restricted to arteries through repression of gene expression in venous endothelial cells by the Notch pathway transcriptional regulator Rbpj. CONCLUSIONS: This study demonstrates a novel mechanism of arterial-venous identity acquisition, indicates a direct link between the Notch and VEGF signaling pathways, and illustrates how cis-regulatory diversity permits differential expression outcomes from a limited repertoire of transcriptional regulators.

The satellite cell in male and female, developing and adult mouse muscle: distinct stem cells for growth and regeneration.

Satellite cells are myogenic cells found between the basal lamina and the sarcolemma of the muscle fibre. Satellite cells are the source of new myofibres; as such, satellite cell transplantation holds promise as a treatment for muscular dystrophies. We have investigated age and sex differences between mouse satellite cells in vitro and assessed the importance of these factors as mediators of donor cell engraftment in an in vivo model of satellite cell transplantation. We found that satellite cell numbers are increased in growing compared to adult and in male compared to female adult mice. We saw no difference in the expression of the myogenic regulatory factors between male and female mice, but distinct profiles were observed according to developmental stage. We show that, in contrast to adult mice, the majority of satellite cells from two week old mice are proliferating to facilitate myofibre growth; however a small proportion of these cells are quiescent and not contributing to this growth programme. Despite observed changes in satellite cell populations, there is no difference in engraftment efficiency either between satellite cells derived from adult or pre-weaned donor mice, male or female donor cells, or between male and female host muscle environments. We suggest there exist two distinct satellite cell populations: one for muscle growth and maintenance and one for muscle regeneration.

Donor satellite cell engraftment is significantly augmented when the host niche is preserved and endogenous satellite cells are incapacitated.

Stem cell transplantation is already in clinical practice for certain genetic diseases and is a promising therapy for dystrophic muscle. We used the mdx mouse model of Duchenne muscular dystrophy to investigate the effect of the host satellite cell niche on the contribution of donor muscle stem cells (satellite cells) to muscle regeneration. We found that incapacitation of the host satellite cells and preservation of the muscle niche promote donor satellite cell contribution to muscle regeneration and functional reconstitution of the satellite cell compartment. But, if the host niche is not promptly refilled, or is filled by competent host satellite cells, it becomes nonfunctional and donor engraftment is negligible. Application of this regimen to aged host muscles also promotes efficient regeneration from aged donor satellite cells. In contrast, if the niche is destroyed, yet host satellite cells remain proliferation-competent, donor-derived engraftment is trivial. Thus preservation of the satellite cell niche, concomitant with functional impairment of the majority of satellite cells within dystrophic human muscles, may improve the efficiency of stem cell therapy.

What is simulated in the action observation network when we observe actions?

In the last decade there has been a great amount of research investigating the role of simulation in our ability to infer the underlying intentions of any observed action. The majority of studies have focussed on the role of mirror neurons and the network of cortical areas active during action observation (AON) in inferring the goal of an observed action. However, it remains unclear what precisely is simulated when we observe an action and how such simulations can enable the observer to infer the underlying intention of that action. In particular it is not known how simulation in the AON enables the inference of the same goal when the kinematics observed to achieve that goal differ, such as when reaching to grasp an object with the left or right hands. Here we performed a behavioural study with healthy human subjects to address this question. We show that the subjects were able to detect very subtle changes in the kinematics of an observed action. In addition, we fitted the behavioural responses with a model based on the predictive coding account of mirror neurons. This is a Bayesian account of action observation that can be explained by the free-energy principle. Here we show that we can model all the effects observed when the action observation system is considered within a predictive coding framework.

Evidence of mirror neurons in human inferior frontal gyrus.

There is much current debate about the existence of mirror neurons in humans. To identify mirror neurons in the inferior frontal gyrus (IFG) of humans, we used a repetition suppression paradigm while measuring neural activity with functional magnetic resonance imaging. Subjects either executed or observed a series of actions. Here we show that in the IFG, responses were suppressed both when an executed action was followed by the same rather than a different observed action and when an observed action was followed by the same rather than a different executed action. This pattern of responses is consistent with that predicted by mirror neurons and is evidence of mirror neurons in the human IFG.