Fig 1.
in vitro PUER cell differentiation reprograms PU.1−/− cells to states resembling in vivo bone marrow macrophage and neutrophil progenitors.
A. Schematic showing the in vitro differentiation of PUER cells into macrophages and neutrophils. Red ticks indicate the timepoints at which cells were sampled for RNA-Seq. B,C The distribution of Euclidean distances between 88h GCSF-OHT samples and other PUER samples or in vivo BM single cells [16] either before (B) or after (C) the removal of batch effects. PUER cells in GCSF and IL3 conditions are labeled as Neu or Mac respectively. BM cells having a population balance analysis (PBA) [18] probability of more than 0.8 for a particular lineage are labeled as that lineage. Ba: basophil, Ery: erythrocyte, Ly: lymphocyte, Mac: monocyte, Neu: granulocyte. BM cells that have PBA probability less than 0.8 for all lineages are labeled as Mixed. The color of the points indicates developmental maturity so that more differentiated cells have higher values. For PUER cells, the developmental maturity is given by the differentiation timepoint scaled to [0,1]. For BM cells, the developmental maturity is given by the additive inverse of the PBA potential scaled to [0,1]. D–G SPRING [19] plots of batch-corrected PUER samples and BM cells. D. Color shows PUER samples in IL3 (macrophage) or GCSF (neutrophil) conditions and BM cells. E. Color annotates PUER samples and BM cells by lineage. Only BM cells having a PBA probability of more than 0.8 for a particular lineage are annotated. F. BM cells are annotated by a heatmap of PBA probability for the monocyte (M) lineage. IL3-OHT PUER cells are annotated by a heatmap of differentiation timepoint. G. BM cells are annotated by a heatmap of PBA probability for the granulocyte (GN) lineage. GCSF-OHT PUER cells are annotated by a heatmap of differentiation timepoint.
Fig 2.
PUER cell differentiation is punctuated by two sharp transitions.
A,B. Pearson correlation coefficient between genome-wide gene expression at each pair of time points in GCSF (A) or IL3 (B) conditions during PUER differentiation (Fig 1A) is plotted as a heatmap. C. Principal components analysis of all the samples. Gene expression was standardized to have zero mean and unit variance. The samples are plotted along the first two principal components, PC1 and PC2, that account for 25% of the total variance.
Fig 3.
The identification of genes expressed differentially between the endpoints of the differentiation.
Gene expressed differentially between the endpoints of PUER differentiation (Fig 1A). A–D. Scatter plots of p-value vs. fold change for all the genes. The p-value and fold change thresholds used to identify DEGs (see Methods) are shown as horizontal and vertical dashed lines respectively. Key differentially expressed lineage markers and TFs have been annotated. E–F. Venn diagrams showing the intersection of the different sets of DEGs identified in this analysis. A. Comparison of undifferentiated PUER cells (−48h) with cells treated with OHT for 7 days in GCSF conditions (168h). B. Comparison of PUER cells pre-treated with GCSF for 48 hours (0h) with cells treated with OHT for 7 days in GCSF conditions (168h). C. Comparison of undifferentiated PUER cells (−48h) with those pre-treated with GCSF for 48 hours (0h). D. Comparison of undifferentiated PUER cells (−48h) with those treated with OHT for 7 days in IL3 conditions (168h). E,F. Overlap of DEGs for selected time points for down-regulated (E) and up-regulated (F) genes.
Table 1.
Summary of biological processes enriched in the top 500 genes for each behavior. The top 30 enriched terms (S26 Fig–S35 Fig) are summarized. Terms in italics were not in the top 30.
Fig 4.
The temporal behaviors of the 10 metagenes identified by NMF.
The behaviors inferred from the genome-wide gene expression time series data (Fig 1A) using NMF are shown in the first two columns. The temporal behavior of each metagene is shown in red. The temporal expression patterns, with maximum expression scaled to 1, of the transcripts having the highest 200 weights for each behavior are shown in black. The last two columns illustrate the reconstruction of the expression pattern of Cx3cr1 by the sequential accumulation of the weighted behaviors , where M is the row number, Hm(t) is the behavior of metagene m, and Wgm is Cx3cr1’s weight for the mth behavior. Cx3cr1’s expression pattern is shown in black and the weighted sum accumulating sequentially from the top to bottom is shown in red.
Fig 5.
Clustering of genome-wide gene expression by temporal behaviors.
The gene expression matrix (Xgt) from the genome-wide time series data (Fig 1A), the temporal behaviors of the 10 metagenes (Hmt)), and the weights of each transcript for each behavior (Wgm) as determined by NMF are depicted on the faces of a rectangular prism. Xgt and Wgm are heatmaps with color representing scaled gene expression or weights respectively. The behaviors are plotted as stacked lineplots with time on the x-axis and expression on the y-axis. Transcripts having the same dominant behavior were grouped together and then sorted in descending order of the weight of the dominant behavior. IL3-OHT and GCSF-OHT samples are shown on the top and bottom prisms respectively.
Fig 6.
The number of DEGs between consecutive time points.
(A) The number of DEGs detected between each pair of consecutive time points (Fig 1A). The overlap between the DEGs detected at different time points in the (B) GCSF or the (C) IL3 conditions.
Fig 7.
GO terms enriched for genes differentially expressed between 0h and 1h or 8h and 12h IL3 samples.
GO terms enriched for DEGs between 0h and 1h and between 8h and 12h are shown on the left and right respectively. The Benjamini-Hochberg adjusted p-value for each enriched GO term is indicated by the color of the point. The number of DEGs overlapping with a term are depicted with the size of the point. The x-axis is the fraction of the DEGs overlapping with a GO term (gene ratio).
Fig 8.
GO terms enriched for genes differentially expressed between 0h and 1h or 8h and 12h GCSF samples.
GO terms enriched for DEGs between 0h and 1h and between 8h and 12h are shown on the left and right respectively. See the legend of Fig 7 for plot description.