Fig. 3: WNT, BMP, and FGF converge into the regulation of DNA replication stress.

a GO term enrichment analysis of common transcripts differentially upregulated by DKK1, FGF2, and Noggin in hiPSCs after 16 h treatment. Expression analyses were performed by single-cell RNA sequencing of >75 cells per condition. After normalizing the data for differences in library size, the ‘FindMarkers’ function with the ROC test was used to determine differentially expressed genes (DEGs) between the different treatment conditions. We selected DEGs with power > 0.25 for the subsequent GO analysis. P-values from GO enrichment analyses are provided directly from DAVID’s EASE score: a one-sided Fisher Exact P-value for gene-enrichment analysis. Data is provided in Supplementary Data 2. b–g DNA combing experiments in hiPSCs treated as indicated for 3 h and labelled with consecutive pulses of CIdU and IdU as shown in (b). Each experiment of every subfigure was replicated at least twice, and the data displayed corresponds to a representative experiment, where is shown the mean ± s.d. of: control (n = 127), Aphidicolin (n = 95), DKK1 (n = 150), FGF2 (n = 135), and Noggin (n = 153) single forks measurements in (c) and control (n = 42), Aphidicolin (n = 59), Aphidicolin + WNT3A (n = 44), Aphidicolin + GSK3i (n = 123) and Aphidicolin + BMP4 (n = 53) single fork measurements in (f). In d, g inter-origin firing distance was assessed. In d control = 46, Aphidicolin = 60, DKK1 = 22, FGF2 = 48, Noggin = 19 inter-origin distances were measured, and in g control (n = 49), Aphidicolin (n = 49), Aphidicolin + WNT3A (n = 31), Aphidicolin + GSK3i (n = 26) and Aphidicolin + BMP4 (n = 59) inter-origin firing distances were measured. In b examples of fork progression and origin (O) are shown. In e a scheme of the replication stress cascade targeted in (f, g) is shown. P-values from one-way ANOVA analyses with multiple comparisons and Dunnet corrections from the indicated groups, ***P < 0.001 or in (f) n.s. > 0.05. h Proposed roles of WNT, BMP, and FGF signalling in DNA replication. i Pipeline for single-cell EdU sequencing. Note that hiPSCs were treated first for 3 h, followed by two 15-min pulses of EdU separated by 1 h. j Replication forks per cell obtained by scEdU-seq in control or DKK1 (3 h) treated hiPSCs from a single biological experiment. Data corresponds to the number of forks per cell in Control (n = 894 cells) and DKK1 (n = 888 cells) after sequencing analysis. Single cells were ranked for their relative position in the S-phase (x-axis) according to their fork distribution pattern across different chromosomes, as previously described⁵⁰. k Replication forks per chromosome per cell, as described in (j). Source data for all experiments are provided as a Source data file, with the exception of (j, k), which is included in the data repository GSE271478. h, i Created with BioRender.com released under a Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International license¹³⁹.