Fig 1.
Chart summarizing experiments and analyses conducted in this study.
Fig 2.
Extent of cardiomyocyte proliferation and differentiation in 2- and 13-day-old mouse hearts.
Immunofluorescent staining showing the presence of proliferating Ki-67+ (A) and mitotic pH3+ (B) cTnT+ cardiomyocytes (yellow arrow heads) at day 2 and day 13. (E) showing the presence of Nkx2.5+ cardiac progenitor cells at day 2 and day 13. Bar charts showing the percentage of cardiomyocytes that are Ki-67+ (C), pH3+ (D) or Nkx2.5+ (F) in 2- and 13-day-old hearts. The data is presented as means ± SD; Scale bar: 50μm, Significantly different: **P<0.01. (G) RT-qPCR assay confirms the immunofluorescent staining that Ki-67+ and pH3 expressions are down-regulated in 13-day-old heart. Relative expression values are presented as means ± SD. Significantly different: *P<0.05, **P<0.01.
Fig 3.
Hierarchical clustering of the microarray results showing the presence of two distinct heart samples.
Relative level of gene expression is color-coded: red implies genes up-regulated in 13-day-old heart while green indicate genes that were down-regulated.
Fig 4.
Gene Ontology enrichment analysis showing representative ontology categories.
(A) Representative down-regulated ontology categories. (B) Representative up-regulated ontology categories. The enrichment p value is displayed on the x-axis.
Fig 5.
KEGG pathway map of the cell cycle.
Cell cycle associated genes are significantly enriched in the list of differentially expressed genes (p = 8.6 x 10−9). Fold change is only shown for genes within the list and illustrates near universal down-regulation in the pathway for cardiomyocytes in the 13-day-old heart, as compared with the 2-day-old heart. Arrows indicate positive regulation, while other lines indicate negative regulation.
Fig 6.
Gene Set Enrichment Analysis (GSEA) of 13-day-old versus 2-day-old mouse hearts.
GSEA analysis reveals that there is significant inhibition of the (A) cell cycle and (B) DNA replication gene sets. On the x-axis, the mouse genes are ranked from the most up-regulated (left end) to the most down-regulated (right end) between 2- and 13-day-old hearts. The “barcode” indicates the position of the genes from the biological pathway. The y-axis shows a running enrichment score (ES), which goes up when genes are encountered in the pathway and goes down otherwise—leading to an assessment of the distribution within the set of all genes. The normalized enrichment score (NES) is inferred from permutations of the gene set and the false discovery rate (FDR). P values are indicated and the plot on the right shows the relative level of gene expression (red = high, blue = low) in the leading edge subset, corresponding to the most significant genes.
Fig 7.
Top gene interaction network based on information from the Ingenuity Pathways Knowledge Base.
Genes that are up- or down-regulated are labelled in red and green, respectively. Genes that are colored gray are not differentially expressed between Day 2 and Day 13.
Fig 8.
Regulator effects network generated from Ingenuity Pathways Analysis.
Upstream regulators are displayed in the top tier, while diseases, functions and phenotypes are displayed in the bottom tier. The dataset genes that connect the upper regulators and the lower diseases and functions are displayed in the middle tier. The causal consistency relationships are represented with orange and blue lines, while the inconsistent relationships are represented as yellow line. Note that some of the dataset genes displayed in the middle tier may have been predicted to be activated or inhibited by upstream regulators, and under such situation is colored orange or blue, respectively (for example CCND1 and E2F1 in the network).
Fig 9.
RT-qPCR validation of representative differentially expressed genes.
Up- (A) and down-regulated (B) in 13-day-old versus 2-day-old mouse hearts. Relative expression values are presented as means ± SD. Significantly different: *P<0.05, **P<0.01, No significance: n.s.