Defining the earliest step of cardiovascular lineage segregation by single-cell RNA-seq

Abstract

Mouse heart development arises from Mesp1-expressing cardiovascular progenitors (CPs) that are specified during gastrulation. The molecular processes that control early regional and lineage segregation of CPs have been unclear. We performed single-cell RNA sequencing of wild-type and Mesp1-null CPs in mice. We showed that populations of Mesp1 CPs are molecularly distinct and span the continuum between epiblast and later mesodermal cells, including hematopoietic progenitors. Single-cell transcriptome analysis of Mesp1-deficient CPs showed that Mesp1 is required for the exit from the pluripotent state and the induction of the cardiovascular gene expression program. We identified distinct populations of Mesp1 CPs that correspond to progenitors committed to different cell lineages and regions of the heart, identifying the molecular features associated with early lineage restriction and regional segregation of the heart at the early stage of mouse gastrulation.

Figure 1:. scRNA-seq of Mesp1+ CPs fills the gap between E6.5 epiblast cells and E7.5 mesodermal cells.

A. Scheme of the experimental strategy used for isolating Mesp1-expressing CPs in vivo. Scale: 200μm. B. SPRING plot of 892 cells showing Mesp1 CPs at E6.75 and E7.25 and the published epiblast cells (E6.5_Scia) and E7.5 Flk1+ progenitors (E7.5_Scia) with read count of Mesp1 > 0. C. SPRING plot coloured by the inferred pseudotime time for all 892 cells.

Figure 2:. Mesp1 controls the exit from pluripotency, EMT and cardiovascular specification.

A. SPRING plot of all 892 cells including Mesp1 KO cells coloured by cell types B. Pseudotime time distribution for WT and Mesp1 KO cells at E6.75. C. Comparison of the genes differentially expressed in scRNA-Seq experiments between control and Mesp1 KO cells and the genes regulated by Mespl gain of function (GOF) in ESC. The 58 genes in agreement with the scRNA-Seq experiment with FDR < 0.1 were highlighted in red. The significance of the overlap was calculated by hypergeometric test using the phyper function in R. D. Gene ontology enrichment for genes downregulated in Mesp1 KO cells. E-G. Violin plots showing the mean and variance difference between WT and Mesp1 KO cells of genes regulating pluripotency (Nanog, Eras) (E), EMT (Cdh1; Snail) (F) and cardiovascular fate (Gata4, Etv2, Myl7) (G).

Figure 3:. Mesp1 single cell analysis identifies different progenitors committed to different fates and heart regions.

A. SPRING analysis of the 807 WT Mesp1-H2B-GFP+ cells at E6.75 and E7.25 and Mesp1+ Scia cells. B. The 5 end points (EPs) revealed by SPRING analysis were considered as 5 distinct cell types (DCT1–5). C. Expression of key genes specific for DCT1–4 D. RNA-FISH of Sox7 and Bmp4 on sections of Mesp1-rtTA/tetO-H2B-GFP embryos at E7.25. E. Heatmap of DCT1 and DCT2 endpoint cells based on unsupervised clustering of the expression of CM and EC marker genes identified at E10.5 (24) combined with newly identified genes enriched in DCT1 or DCT2. F-G. RNA-FISH of Foxc2 and Wnt2b on sections of Mesp1-rtTA/tetO-H2B-GFP embryos at E7.25. Higher magnification is found in G. Scale: 50 μm.

Figure 4:. Notch1 marks Mesp1 progenitors committed to the endocardial fate.

A. Violin plot of Notch1 expression in DCT1 and DCT2 cells. B. Notch1 RNA-FISH on a section of an E7.25 Mesp1-rtTA/tetO-H2B-GFP embryo. scale: 50 μm. C. Experimental strategy used for tracing Notch1 expressing cells at E6.5. D-G. Confocal analysis of immunostaining for endoglin (EC marker) (D-F) and cTNT (CM marker) (G) of Notch1-CreERT2/Rosa-tdTomato heart sections at E12.5. Lower magnification (D) Scale: 500 μm. Higher magnifications of the LV (E) and RA (G) showed that most tdTomato + cells are ECs although rare CMs are also marked (asterisks). Scale: 200 μm. H, I. Percentage of tdTomato positive cells in ECs, CMs and EPs in the ventricles (H) (7804 cells counted; n=6 embryos from 3 different litters) and in the atria (I) (4819 cells counted; n= 7 embryos from 3 different litters).