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

<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

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

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

Existence of a positive feedback loop to activate the <i>Tbx5</i> gene.

<p>(A) An sgRNA was designed to target the sequence (shown in red) immediately downstream of the first methionine codon (ATG in blue). The underlined TGG sequence corresponds to the protospacer adjacent motif (PAM). (B) Obtained internal deletion alleles in a <i>Tbx5</i> null ES cell line. The first methionine codon (ATG) is shown in bold, and the PAM (TGG) is shown in green. (C) Immunoblot analysis of TBX5 and eYFP in differentiated BAC <i>Tbx5</i>^CreERT2/<i>ROSA26</i>^eYFP/eYFP ES cells that were either wild type or null (k/o) for <i>Tbx5</i>. α-Tubulin was examined as a loading control. The whole blotted membrane is indicated as a whole in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0140831#pone.0140831.s009" target="_blank">S8 Fig</a>. (D) BAC <i>Tbx5</i>^CreERT2<i>/ROSA26</i>^eYFP/eYFP ES cells either rendered <i>Tbx5</i> null by the CRISPR/Cas9 or left unmodified (WT) were induced to differentiate into cardiomyocytes in the presence of 4-hydroxytamoxifen. The cells were then subjected to flow cytometric analysis of TNNT2 and eYFP expression. ES cells without the BAC transgene were used as control. (E) Representative flow cytometric plots as well as mean ± SEM values from three independent experiments are shown. *<i>P</i> < 0.05; NS, not significant (Student's <i>t</i> test). (F) Schematic representation of the early CPs. CMs; cardiomyocytes, CSCs; electric conduction system cells.</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

Single-cell expression profiling of the earliest CPs.

<p>(A) WISH analysis of <i>Nkx2-5</i> and <i>Tbx5</i> expression in mouse embryos at EB, late bud (LB), and EHF stages. Embryos are shown in left lateral view. A; anterior, P; posterior, Red arrows; the most anterior part of the embryo. (B) Strategy for generation of single-cell cDNA preparations. (C) Classification of single-cell cDNA preparations of CPs from EB, LB, and EHF stages by PCR analysis of marker genes. The number of preparations is shown in parentheses. (D) Subpopulation shift between EB and Somite stages. (E) Taqman assay for <i>Nkx2-5</i> and <i>Tbx5</i> on constructed single-cell cDNA preparations. (F) Distribution of <i>Nkx2-5</i>-expressing CPs and <i>Tbx5</i>-expressing CPs in EHF stage embryo. The pictures of the embryos in the upper panel indicate whole mount <i>in situ</i> hybridization for <i>Nkx2-5</i> and <i>Tbx5</i> in EHF stage embryos in the frontal view. Note the area of <i>Nkx2-5</i> is wider than that of <i>Tbx5</i>. Fluorescence images indicate the immunostained EHF stage mouse embryo for NKX2-5 and TBX5. The illustration at the upper right panel shows section plane of fluorescence image. D; distal, EN; endoderm, NE; neural ectoderm. Blue; 4ʹ,6-diamidino-2-phenylindole (DAPI), Green; TBX5, Red; NKX2-5, L; left, P; proximal, R; right. Scale bar; 100 μm.</p

The production of <i>Tbx5</i>-expressing CPs terminates by the EHF stage.

<p>(A) Horizontal section of a BAC <i>Tbx5</i>^CreERT2/<i>ROSA26</i>^eYFP/eYFP mouse embryo at the somite stage stained for TBX5 (red) and eYFP (green) is indicated in the right panel. This embryo was dissected at EB stage, pulse-labelled via transient exposure to 4-hydroxytamoxifen <i>in vitro</i> for 3 hours followed by the withdrawal of tamoxifen, and cultured for 24 hours via whole embryonic culture without 4-hydroxytamoxifen up to the early Somite stage. Red arrows indicate <i>Tbx5</i>-expressing cells labelled at the EB stage, and asterisks indicate the background signal in the endoderm due to the anti-mouse secondary antibodies on mouse section. Data are representative of five embryos. Left panel indicates the illustration of experimental design. hf, headfold. Scale bar, 50 μm. (B) Quantitative cytometric plot of the pulse-labelling experiment presented in C and E is indicated as mean ± SEM values. *<i>P</i><0.05. (C) BAC <i>Tbx5</i>^CreERT2/<i>ROSA26</i>^eYFP/eYFP embryos were pulse-labelled by transient exposure to 4-hydroxytamoxifen at the LB stage, and a tissue fragment from the anterior portion of each embryo was then cultured in the absence of 4-hydroxytamoxifen for 6 days before confocal immunofluorescence analysis with antibodies to TBX5 and to eYFP. Data are representative of four embryos. Blue fluorescence; DAPI, White arrowheads; TBX5⁺/eYFP⁻cells. Scale bar, 100 μm. (D) Schematic representation of CP eYFP^-/TBX5⁺ progeny. (E) Embryos were analyzed as in (C) with the exception that pulse-labelling was performed at the EHF stage. Data are representative of six embryos. TBX5⁺/eYFP⁻cells were largely absent. Scale bar, 100 μm. (F) Schematic explanation of CP eYFP⁺/TBX5⁺-only progeny. A CP source producing TBX5⁺ cells is not present after pulse-labelling.</p