Inhibition of FGF and TGF-β Pathways in hESCs Identify STOX2 as a Novel SMAD2/4 Cofactor

Simple Summary Signaling pathways are the means by which cells and tissue communicate, orchestrating key events during mammalian development, homeostasis, and disease. During development, signaling determines the identity of cells, and thereby controls morphogenesis and organ specification. Depending on the cellular context, these pathways can exert a broad range of even opposing functions. This is achieved, among other mechanisms, by crosstalk between pathways. Here, we examined how two pathways (the transforming growth factor-beta (TGF-beta) and the fibroblast growth factor (FGF)) cooperate in the maintenance and cell fate specification of human embryonic stem cells. We used inhibitory molecules for individual pathways on a short time series and analyzed the resulting variation in gene expression. In contrast to our expectations, we did not observe an extended crosstalk between the pathway at the gene regulatory level. However, we discovered STOX2 as a new primary target of the TGF-beta signaling pathway. Our results show that STOX2 might act as a novel TGF-beta signaling co-factor. Our work will contribute to understand how signaling by the TGF-beta is mediated. In the future, these results might help to deepen our understanding of how signaling is propagated. The fibroblast growth factor (FGF) and the transforming growth factor-beta (TGF-beta) pathways are both involved in the maintenance of human embryonic stem cells (hESCs) and regulate the onset of their differentiation. Their converging functions have suggested that these pathways might share a wide range of overlapping targets. Published studies have focused on the long-term effects (24-48 h) of FGF and TGF-beta inhibition in hESCs, identifying direct and indirect target genes. In this study, we focused on the earliest transcriptome changes occurring between 3 and 9 h after FGF and TGF-beta inhibition to identify direct target genes only. Our analysis clearly shows that only a handful of target transcripts are common to both pathways. This is surprising in light of the previous literature, and has implications for models of cell signaling in human pluripotent cells. In addition, we identified STOX2 as a novel primary target of the TGF-beta signaling pathway. We show that STOX2 might act as a novel SMAD2/4 cofactor. Taken together, our results provide insights into the effect of cell signaling on the transcription profile of human pluripotent cells