Population-level antagonism between FGF and BMP signaling steers mesoderm differentiation in embryonic stem cells. Population-level antagonism between FGF and BMP signaling steers mesoderm differentiation in embryonic stem cells

The mesodermal precursor populations for different internal organ systems are specified during gastrulation by the combined activity of extracellular signaling systems such as BMP, Wnt, Nodal, and FGF. The BMP, Wnt and Nodal signaling requirements for the differentiation of specific mesoderm subtypes in mammals have been mapped in detail, but which mesodermal cell types depend on FGF signaling is not precisely known. It is also not clear how FGF signaling modulates the activity of orthogonal signaling systems involved in mesoderm differentiation. Here, we address these questions by analyzing the effects of targeted signaling manipulations in differentiating stem cell populations with single cell resolution. We identify opposing functions of BMP and FGF, and map the boundary between FGF-dependent and -independent mesodermal lineages. Stimulation with exogenous FGF boosts the expression of endogenous Fgfs while repressing Bmp ligands. This intercellular positive autoregulation of FGF signaling coupled to the repression of BMP signaling may contribute to the specification of reproducible and coherent cohorts of cells with the same identity via a community effect, both in the embryo and in synthetic embryo-like systems. Overall design: Mouse epiblast stem cells were differentiated towards a mix of mesodermal cell types in N2B27 medium supplemented with BMP4, CHIR99201 and different doses of FGF2 or the FGF receptor inhibitor AZD4547, and analyzed by scRNAseq.

不同内脏器官系统的中胚层前体细胞群，在原肠胚形成阶段由BMP、Wnt、Nodal以及成纤维细胞生长因子（FGF）等细胞外信号通路的协同活性所特化。目前已详细阐明了哺乳动物中特定中胚层亚型分化所需的BMP、Wnt及Nodal信号通路需求，但尚不清楚哪些中胚层细胞类型依赖于FGF信号通路。此外，目前也尚不明确FGF信号通路如何调控参与中胚层分化的正交信号系统的活性。本研究通过以单细胞分辨率分析靶向信号操控对分化中干细胞群的影响，解决了上述问题。本研究揭示了BMP与FGF的拮抗功能，并绘制了依赖FGF与非依赖FGF的中胚层细胞谱系之间的边界。外源性FGF刺激可增强内源性Fgfs的表达，同时抑制Bmp配体的表达。这种FGF信号通路的细胞间正向自调控，与BMP信号通路的抑制相协同，可能通过群落效应，在胚胎以及类合成胚胎系统中，促成具有相同细胞身份的、可重复且均一的细胞群的特化。实验整体设计：将小鼠上胚层干细胞在添加了BMP4、CHIR99201以及不同剂量FGF2或FGF受体抑制剂AZD4547的N2B27培养基中，向混合的中胚层细胞类型进行分化，并通过单细胞RNA测序（scRNAseq）进行分析。