Single-Cell Expression Profiling Reveals a Dynamic State of Cardiac Precursor Cells in the Early Mouse Embryo

In the early vertebrate embryo, cardiac progenitor/precursor cells (CPs) give rise to cardiac structures. Better understanding their biological character is critical to understand the heart development and to apply CPs for the clinical arena. However, our knowledge remains incomplete. With the use of single-cell expression profiling, we have now revealed rapid and dynamic changes in gene expression profiles of the embryonic CPs during the early phase after their segregation from the cardiac mesoderm. Progressively, the nascent mesodermal gene Mesp1 terminated, and Nkx2-5+/Tbx5+ population rapidly replaced the Tbx5low+ population as the expression of the cardiac genes Tbx5 and Nkx2-5 increased. At the Early Headfold stage, Tbx5-expressing CPs gradually showed a unique molecular signature with signs of cardiomyocyte differentiation. Lineage-tracing revealed a developmentally distinct characteristic of this population. They underwent progressive differentiation only towards the cardiomyocyte lineage corresponding to the first heart field rather than being maintained as a progenitor pool. More importantly, Tbx5 likely plays an important role in a transcriptional network to regulate the distinct character of the FHF via a positive feedback loop to activate the robust expression of Tbx5 in CPs. These data expands our knowledge on the behavior of CPs during the early phase of cardiac development, subsequently providing a platform for further study

Studying the neural mechanisms augmenting the neurohumoral drive in chronic heart failure

EThOS - Electronic Theses Online ServiceGBUnited Kingdo

Arterial pressure and gene expression in the nucleus of the solitary tract in rats fed with high-fat diet

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES

Cardiovascular effects of chronically increasing angiotensin II type 2 receptor expression in the nucleus of the solitary tract

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES

Importance of All and AT2 receptors in the nucleus of the solitary tract in cardiovascular responses induced by a high-fat diet

A high-fat diet (HFD) induces an increase in arterial pressure and a decrease in baroreflex function, which may be associated with increased expression of angiotensin type 1 receptor (AT1R) and pro-inflammatory cytokine genes and reduced expression of the angiotensin type 2 receptor (AT2R) gene within the nucleus of the solitary tract (NTS), a key area of the brainstem involved in cardiovascular control. Thus, in the present study, we evaluated the changes in arterial pressure and gene expression of components of the renin-angiotensin system (RAS) and neuroinflammatory markers in the NTS of rats fed a HFD and treated with either an AT1R blocker or with virus-mediated AT2R overexpression in the NTS. Male Holtzman rats (300-320 g) were fed either a standard rat chow diet (SD) or HFD for 6 weeks before commencing the tests. AT1R blockade in the NTS of HFD-fed rats attenuated the increase in arterial pressure and the impairment of reflex bradycardia, whereas AT2R overexpression in the NTS only improved the baroreflex function. The RFD also increased the hypertensive and decreased the protective axis of the RAS and was associated with neuroinflammation within the NTS. The expression of angiotensin-converting enzyme and neuroinflammatory components, but not AT1R, in the NTS was reduced by AT2R overexpression in this site. Based on these data, AT1R and AT2R in the NTS are differentially involved in the cardiovascular changes induced by a HFD. Chronic inflammation and changes in the RAS in the NTS may also account for the cardiovascular responses observed in HFD-fed rats424439449CONSELHO NACIONAL DE DESENVOLVIMENTO CIENTÍFICO E TECNOLÓGICO - CNPQCOORDENAÇÃO DE APERFEIÇOAMENTO DE PESSOAL DE NÍVEL SUPERIOR - CAPESFUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULO - FAPESP304873/2014-4; 425586/2016-2; 308099/2017-633001014037P42013/13118-0; 2015/234677The authors thank Dr. Rodrigo Tomeo and Dr. Regina C. Vendramini for providing expert technical assistance, Silvana A.D. Malavolta and Carla D. Alencar for providing secretarial assistance and Ana V. Oliveira for providing animal care. This study was supported by Fundação de Amparo à Pesquisa do Estado de São Paulo [2013/13118-0; 2015/234677]; Conselho Nacional de Desenvolvimento Científico e Tecnológico [304873/2014-4; 425586/2016-2; 308099/2017-6]; Coordenação de Aperfeiçoamento de Pessoal de Nível Superior [33001014037P4 and Finance Code 001]; and NIH grant HL-07803

Distinct molecular signature of <i>Nkx2-5</i>⁺/<i>Tbx5</i>⁺ CPs at the EHF Stage.

<p>(A) PCA for the results of deep sequencing of single-cell cDNA preparations from cells of the indicated subpopulations. Circles of lighter colour represent each of the cell subpopulations, and those of darker colour represent the centroid for each cell subpopulation. Each ellipse indicates the standard deviation. (B) Heat-map of the expression of enriched key genes in each subpopulation. <i>Nkx2-5</i> and <i>Mef2c</i> were not enriched in any subpopulation. The intensity was calculated by the formula; z = (x-μ)/σ. z; intensity, x; value of Reads per Million, μ average, σ standard deviation. (C) Top ten categories of GO enrichment analysis in each subpopulation.</p

<i>Tbx5</i>-Expressing FHF cells derived from ES cells are unipotent.

<p>(A) Alkaline phosphatase staining for BAC <i>Tbx5</i>^CreERT2/<i>ROSA26</i>^eYFP/eYFP ES cells. Scale bar, 100 μm. (B) BAC <i>Tbx5</i>^CreERT2/<i>ROSA26</i>^eYFP/eYFP ES cells stained with SSEA1 antibody. Blue; DAPI. Scale bar, 100 μm. (C) RT-PCR analysis of <i>Oct3/4</i> and <i>Sox2</i> expression in BAC <i>Tbx5</i>^CreERT2/<i>ROSA26</i>^eYFP/eYFP ES cells. (D) Genomic PCR analysis for <i>CreERT2</i> and <i>Sry</i> in isolated BAC <i>Tbx5</i>^CreERT2/<i>ROSA26</i>^eYFP/eYFP ES cell lines. Only those with a male karyotype (<i>Sry</i> positive) were chosen for further experiments. Clones #2 and #3 are female and male, respectively. (E) BAC <i>Tbx5</i>^CreERT2<i>/ROSA26</i>^eYFP/eYFP ES cells were induced to differentiate into cardiomyocytes <i>in vitro</i> in the presence of 4-hydroxytamoxifen. The cells at differentiation day 14 were then stained with antibodies to eYFP as well as with those to TNNT2, HCN4, PECAM1, or ACTA2A. Blue; DAPI. Scale bars, 100 μm.</p

The FHF CPs marked by BAC <i>Tbx5</i>^CreERT2 transgene are unipotent cells that contribute only to the cardiomyocyte lineage.

<p>(A-G). Sections of the heart of E15.5 BAC <i>Tbx5</i>^CreERT2/<i>ROSA26</i>^eYFP/eYFP mouse embryos treated with tamoxifen at E7.5 <i>in vivo</i> were subjected to confocal immunofluorescence analysis with antibodies to the indicated proteins. Nuclei were stained with DAPI. The boxed region in (A) is shown at higher magnification in (D), and those in (B) are shown at higher magnification in (F) and (G). Data are representative of three embryos. Asterisks indicate the sinoatrial node (F) or the bundle of His (G). The white arrow in (G) indicates the AV node. (H) Horizontal section stained with indicated antibodies at the atrial level of a BAC <i>Tbx5</i>^CreERT2/<i>ROSA26</i>^eYFP/eYFP mouse embryo at E10.5 after tamoxifen treatment at E7.5 <i>in vivo</i>. TBX5⁺ cells in the DMP (white arrows) were negative for eYFP. LA; left atrium, RA; right atrium, RAVC; right anterior vena cava. Scale bars: 100 μm (A and B), 20 μm (C–G) and 200 μm (H).</p

Dynamic Changes in the Expression Profiles of CPs from the EB to EHF Stages.

<p>(A) Proportion of single-cell cDNA preparations positive for <i>Mesp1</i>, <i>Myl2</i>, <i>Isl1</i>, and <i>Myl7</i> expression at the indicated embryonic stages as determined by PCR analysis. (B) WISH analysis of <i>Mesp1</i>, <i>Myl2</i>, <i>Isl1</i>, and <i>Myl7</i> expression in the mouse embryo at the EB, LB, and EHF stages. Embryos are shown in the left lateral view. Expression of <i>Mesp1</i> was detected at a low level in the anterior mesoderm at the EB stage. <i>Myl2</i> was not detected as expected by PCR analysis on single cell cDNA preparations in (A). A; anterior, P; posterior.</p