Probing cell identity hierarchies by fate titration and collision during direct reprogramming (2022)
Figure 5

Figure 5: Fate competition between the bHLH factors MyoD1 and Ascl1

A. Louvain clustering superimposed on a Uniform Manifold Approximation and Projection (UMAP) embedding of the second Collide-seq experiment. Clusters are labelled according to the predominant transcription factor or cell state.

B. Condition based RNA velocity and CellRank for the indicated conditions. Shown are UMAP embeddings of all cells in the dataset (grey) with cells positive for the indicated transcription factor(s) colored by the terminal fate assigned by CellRank to indicate lineage endpoints (Bergen et al, 2020). See RNA velocity and CellRank analysis section of Methods for further details.

C. Intersections of genes bound by Ascl1 and/or MyoD1 based on ChIP-seq data (Lee et al, 2020) (see ChIP-seq data analysis and synergism and antagonism annotation sections in Methods for further details) with gene sets that correspond to synergistic and antagonistic up- and down-regulation. We defined synergism and antagonism based on differential expression analysis (See differential expression analysis section in Methods for further details). Genes were considered synergistic or antagonistic when their expression in double positive conditions was higher (synergism) or lower (antagonism) than expected by adding their expression in the single factor conditions together. Shown is the size of the intersection (number of genes) and the relative size of this intersection with respect to the respective differentially expressed gene sets as defined by the column.

D. Log-normalized expression z-score of genes up- (top panels) and downregulated (bottom panels) by Ascl1 (left, blue violins) and MyoD1 (right, yellow violins) versus their scaled expression levels (x-axis) binned into 10% intervals. For each box, the centerline defines the median, the height of the box is given by the interquartile range (IQR), the whiskers are given by 1.5 * IQR and outliers are given as points beyond the minimum or maximum whisker.

E. Fate titration plot of Ascl1 and MyoD1 double positive cells (see fate titration section in Methods for further details). Shown are decision boundaries for indicated fates according to transcription factor levels. MyoD1 and Ascl1 expression shown on the x- and y-axis are log-normalized expression values scaled into the dynamic range of the single-positive condition (see fate titration analysis section in Methods for further details). Cells are colored according to their lineage endpoint as determined by CellRank.

F. Representative immunofluorescence images of cells 3 days after transgene induction. Cells were stained for the fluorescent reporter present on the PiggyBac construct of the reprogramming factor. Arrows indicate multinucleated cells. Scale bar represents 50 µm. mutAscl1 = mutant Ascl1.

G. Quantification of the percentage of cells with more than one nucleus among all transfected cells within a given condition. Data points represent biological replicates (n = 4). For each box, the centerline defines the median, the height of the box is given by the interquartile range (IQR), the whiskers are given by 1.5 * IQR and outliers are given as points beyond the minimum or maximum whisker. Pairwise comparisons performed with Mann-Whitney U test, correction for multiple testing performed with Benjamini-Hochberg correction. *: p < 0.05 (MyoD1 vs Ascl1: p = 0.03, MyoD1 vs mutAscl1: p = 0.03, MyoD1 vs Ascl1 & MyoD1: p = 0.03, MyoD1 vs mutAscl1 & MyoD1: p = 0.03, Ascl1 vs Ascl1 & MyoD1: p = 0.03, Ascl1 vs mutAscl1 & MyoD1: p = 0.03, mutAscl1 vs Ascl1 & MyoD1: p = 0.03, mutAscl1 vs mutAscl1 & MyoD1: p = 0.03)