Fig 1.
Schematic overview and FACS analysis of in vitro, murine Nkx2.5 EmGFP ES cell-based cardiac differentiation.
A) schematic representation of homologously recombineered, 2A self-cleaving EmGFP reporter inserted immediately downstream of the endogenous Nkx2.5 promoter and immediately upstream of the Nkx2.5 gene in mouse ES cells, B) overview of gene expression trends for in vitro cardiac-directed differentiation, sorting and purification of Nkx2.5 EmGFP reporter ES cells, C) FACS analysis of Nkx2.5 EmGFP ES cells at d6, d8 and d10 of cardiac differentiation, D) EmGFP gene expression kinetics for non-sorted Nkx2.5 EmGFP ES cells throughout cardiac differentiation (* p<0.05, n = 3).
Fig 2.
Gene expression dynamics for pluripotency-, mesoderm- and cardiac lineage-associated transcription factors in cardiac-differentiated Nkx2.5 EmGFP ES cells.
A) pluripotency markers Nanog, Oct4 and Sox2, B) mesodermal marker Brachyury T and early cardiac lineage transcription factors Mesp1, Nkx2.5, Tbx5 and Isl1, C) cardiac-specific contractile proteins Myh6 and Tnnt2 (*p<0.05 vs. d0 undifferentiated; n = 3).
Fig 3.
RNA-Seq data for d8 and CLK CPCs confirms enrichment for protein-coding and lncRNA genes associated with CPCs as well as the distribution of differentially-expressed annotated and unannotated genes.
A) RNA-seq-derived RPKM values for pluripotency and cardiac-related genes as proportionally represented by d8/CLK CPCs and undifferentiated ES cells, B) distribution of RPKM values for d8/CLK CPCs and undifferentiated ES cells with respect to known cardiac-related lncRNAs, C) graphical summary of d8/CLK enrichment for panel of lncRNA genes, previously identified by Wamstead et al, in cardiac-differentiated ES cells, D) Biaxial Venn plot showing distribution of differentially-expressed annotated and unannotated genes in d8/CLK CPCs relative to undifferentiated ES cells, highlighting transcripts that are commonly up- and down-regulated.
Fig 4.
Sashimi read density plots show unannotated, putative lncRNAs common to d8/CLK CPCs that are differentially expressed relative to undifferentiated ES cells and map adjacent to cardiac-related protein-coding genes.
A) 247nt transcript commonly upregulated in d8/CLK CPCs, approximately 1Kb upstream of Adamts6, B) 2,024nt transcript upregulated in d8/CLK CPCs, immediately upstream of Ctdspl, C) 1,378nt transcript downregulated in d8/CLK CPCs, upstream of Ccnb1ip1.