Antoine Bondue

Mesp1 Acts as a Master Regulator of Multipotent Cardiovascular Progenitor Specification

During embryonic development, multipotent cardiovascular progenitor cells are specified from early mesoderm. Using mouse ESCs in which gene expression can be temporally regulated, we have found that transient expression of Mesp1 dramatically accelerates and enhances multipotent cardiovascular progenitor specification through an intrinsic and cell autonomous mechanism. Genome-wide transcriptional analysis indicates that Mesp1 rapidly activates and represses a discrete set of genes, and chromatin immunoprecipitation shows that Mesp1 directly binds to regulatory DNA sequences located in the promoter of many key genes in the core cardiac transcriptional machinery, resulting in their rapid upregulation. Mesp1 also directly represses the expression of key genes regulating other early mesoderm and endoderm cell fates. Our results demonstrate that Mesp1 acts as a key regulatory switch during cardiovascular specification, residing at the top of the hierarchy of the gene network responsible for cardiovascular cell-fate determination.

The Core Domain of Chemokines Binds CCR5 Extracellular Domains while Their Amino Terminus Interacts with the Transmembrane Helix Bundle

CCR5 is a functional receptor for various inflammatory CC-chemokines, including macrophage inflammatory protein (MIP)-1α and RANTES (regulated on activation normal T cell expressed and secreted), and is the main coreceptor of human immunodeficiency viruses. The second extracellular loop and amino-terminal domain of CCR5 are critical for chemokine binding, whereas the transmembrane helix bundle is involved in receptor activation. Chemokine domains and residues important for CCR5 binding and/or activation have also been identified. However, the precise way by which chemokines interact with and activate CCR5 is presently unknown. In this study, we have compared the binding and functional properties of chemokine variants onto wild-type CCR5 and CCR5 point mutants. Several mutations in CCR5 extracellular domains (E172A, R168A, K191A, and D276A) strongly affected MIP-1α binding but had little effect on RANTES binding. However, a MIP/RANTES chimera, containing the MIP-1α N terminus and the RANTES core, bound to these mutants with an affinity similar to that of RANTES. Several CCR5 mutants affecting transmembrane helices 2 and 3 (L104F, L104F/F109H/F112Y, F85L/L104F) reduced the potency of MIP-1α by 10–100 fold with little effect on activation by RANTES. However, the MIP/RANTES chimera activated these mutants with a potency similar to that of MIP-1α. In contrast, LD78β, a natural MIP-1α variant, which, like RANTES, contains a proline at position 2, activated these mutants as well as RANTES. Altogether, these results suggest that the core domains of MIP-1α and RANTES bind distinct residues in CCR5 extracellular domains, whereas the N terminus of chemokines mediates receptor activation by interacting with the transmembrane helix bundle.

Mesp1: A Key Regulator of Cardiovascular Lineage Commitment

In mammals, the heart arises from the differentiation of 2 sources of multipotent cardiovascular progenitors (MCPs). Different studies indicated that an evolutionary conserved transcriptional regulatory network controls cardiovascular development from flies to humans. Whereas in Drosophila, Tinman acts as a master regulator of cardiac development, the identification of such a master regulator in mammals remained elusive for a long time. In this review, we discuss the recent findings suggesting that Mesp1 acts as a key regulator of cardiovascular progenitors in vertebrates. Lineage tracing in mice demonstrated that Mesp1 represents the earliest marker of cardiovascular progenitors, tracing almost all the cells of the heart including derivatives of the primary and second heart fields. The inactivation of Mesp1/2 indicated that Mesp genes are essential for early cardiac mesoderm formation and MCP migration. Several recent studies have demonstrated that Mesp1 massively promotes cardiovascular differentiation during embryonic development and pluripotent stem cell differentiation and indicated that Mesp1 resides at the top of the cellular and transcriptional hierarchy that orchestrates MCP specification. In primitive chordates, Mesp also controls early cardiac progenitor specification and migration, suggesting that Mesp arises during chordate evolution to regulate the earliest step of cardiovascular development. Defining how Mesp1 regulates the earliest step of MCP specification and controls their migration is essential to understand the root of cardiovascular development and how the deregulation of these processes can lead to congenital heart diseases. In addition, these findings will be very useful to boost the production of cardiovascular cells for cellular therapy, drug and toxicity screening.

Defining the earliest step of cardiovascular progenitor specification during embryonic stem cell differentiation

Mesp1, the earliest marker of cardiovascular development in vivo, is used to isolate and characterize multipotent cardiovascular progenitors during ESC differentiation.

Early lineage restriction in temporally distinct populations of Mesp1 progenitors during mammalian heart development

Cardiac development arises from two sources of mesoderm progenitors, the first heart field (FHF) and the second (SHF). Mesp1 has been proposed to mark the most primitive multipotent cardiac progenitors common for both heart fields. Here, using clonal analysis of the earliest prospective cardiovascular progenitors in a temporally controlled manner during early gastrulation, we found that Mesp1 progenitors consist of two temporally distinct pools of progenitors restricted to either the FHF or the SHF. FHF progenitors were unipotent, whereas SHF progenitors were either unipotent or bipotent. Microarray and single-cell PCR with reverse transcription analysis of Mesp1 progenitors revealed the existence of molecularly distinct populations of Mesp1 progenitors, consistent with their lineage and regional contribution. Together, these results provide evidence that heart development arises from distinct populations of unipotent and bipotent cardiac progenitors that independently express Mesp1 at different time points during their specification, revealing that the regional segregation and lineage restriction of cardiac progenitors occur very early during gastrulation.

The expression of Sox17 identifies and regulates haemogenic endothelium

Although it is well recognized that haematopoietic stem cells (HSCs) develop from a specialized population of endothelial cells known as haemogenic endothelium, the regulatory pathways that control this transition are not well defined. Here we identify Sox17 as a key regulator of haemogenic endothelial development. Analysis of Sox17–GFP reporter mice revealed that Sox17 is expressed in haemogenic endothelium and emerging HSCs and that it is required for HSC development. Using the mouse embryonic stem cell differentiation model, we show that Sox17 is also expressed in haemogenic endothelium generated in vitro and that it plays a pivotal role in the development and/or expansion of haemogenic endothelium through the Notch signalling pathway. Taken together, these findings position Sox17 as a key regulator of haemogenic endothelial and haematopoietic development.

Eomesodermin induces Mesp1 expression and cardiac differentiation from embryonic stem cells in the absence of Activin

The transcription factor Eomesodermin (Eomes) is involved in early embryonic patterning, but the range of cell fates that it controls as well as its mechanisms of action remain unclear. Here we show that transient expression of Eomes promotes cardiovascular fate during embryonic stem cell differentiation. Eomes also rapidly induces the expression of Mesp1, a key regulator of cardiovascular differentiation, and directly binds to regulatory sequences of Mesp1. Eomes effects are strikingly modulated by Activin signalling: high levels of Activin inhibit the promotion of cardiac mesoderm by Eomes, while they enhance Eomes‐dependent endodermal specification. These results place Eomes upstream of the Mesp1‐dependent programme of cardiogenesis, and at the intersection of mesodermal and endodermal specification, depending on the levels of Activin/Nodal signalling.

Mesp1 controls the speed, polarity, and directionality of cardiovascular progenitor migration

The transcription factors Mesp1 and Mesp2 are equally efficient at promoting specification, EMT, and differentiation of early multipotent cardiovascular progenitors. However, only Mesp1 promotes the speed, polarity, and directionality of cell migration, explaining how Mesp1 coordinates progenitor fate decision and migration during development.

Pulmonary hypertension and ventilation during exercise: Role of the pre-capillary component

BACKGROUND Excessive exercise-induced hyperventilation and high prevalence of exercise oscillatory breathing (EOB) are present in patients with post-capillary pulmonary hypertension (PH) complicating left heart disease (LHD). Patients with pre-capillary PH have even higher hyperventilation but no EOB. We sought to determine the impact of a pre-capillary component of PH on ventilatory response to exercise in patients with PH and left heart disease. METHODS We retrospectively compared patients with idiopathic or heritable pulmonary arterial hypertension (PAH, n = 29), isolated post-capillary PH (IpcPH, n = 29), and combined post- and pre-capillary PH (CpcPH, n = 12). Diastolic pressure gradient (DPG = diastolic pulmonary artery pressure - pulmonary wedge pressure) was used to distinguish IpcPH (DPG <7 mm Hg) from CpcPH (DPG ≥7 mm Hg). RESULTS Pulmonary vascular resistance (PVR) was higher in PAH, intermediate in CpcPH, and low in IpcPH. All patients with CpcPH but 1 had PVR >3 Wood unit. Exercise-induced hyperventilation (high minute ventilation over carbon dioxide production, low end-tidal carbon dioxide) was marked in PAH, intermediate in CpcPH, and low in IpcPH (p < 0.001) and correlated with DPG and PVR. Prevalence of EOB decreased from IpcPH to CpcPH to PAH (p < 0.001). CONCLUSIONS Patients with CpcPH may have worse hemodynamics than patients with IpcPH and distinct alterations of ventilatory control, consistent with more exercise-induced hyperventilation and less EOB. This might be explained at least in part by the presence and extent of pulmonary vascular disease.

Characterization of a novel VPAC1 selective agonist and identification of the receptor domains implicated in the carboxyl-terminal peptide recognition

Vasoactive Intestinal Polypeptide (VIP) interacts with a high affinity to two subclasses of G protein coupled receptors named VPAC1 and VPAC2, and has a 3–10 fold preference for VPAC1 over VPAC2 receptors. Selective ligands for each receptor subclass were recently described. [R16]‐PACAP (1–23) and [L22]‐VIP are two selective VPAC1 agonists. Chimaeric human VPAC2‐VPAC1 recombinant receptors expressed in CHO cells were used to identify the receptor domains implicated in these two selective ligands recognition. The VPAC2 preference for [R16]‐PACAP (1–27) over [R16]‐PACAP (1–23) did not require the receptors NH2‐terminus domain but involved the whole transmembrane domain. In contrast, the selectivity of [L22]‐VIP depended only on the presence of the NH2 terminus and EC2 domains of the VPAC1 receptor. The present data support the idea that in the GPCR‐B family of receptors the different selective ligands require different domains for their selectivity, and that the peptides carboxyl terminal sequence (amino acids 24–27) folds back on the transmembrane receptor domain, close to the peptides, aminoterminus.