Developmental signals control chromosome segregation fidelity during early lineage specification and neurogenesis by modulating replicative stress. Developmental signals control chromosome segregation fidelity during early lineage specification and neurogenesis by modulating replicative stress

Human development and homeostasis rely on the correct replication, maintenance and segregation of our genetic blueprints. How these intracellular processes are monitored across different human cellular lineages, and why the spatio-temporal distribution of mosaicism varies during development remain unknown. Using human and mouse pluripotent stem cells, we identify that several lineage specification signals –including WNT, BMP and FGF– converge into the modulation of DNA replication stress and damage during S-phase, which in turn controls spindle dynamics and chromosome segregation fidelity in mitosis. We show that patterning signals associated with anteriorisation during mammalian gastrulation increase chromosome missegregation, while the posteriorising signals WNT and BMP protect pluripotent stem cells from excessive origin firing, DNA damage and chromosome missegregation derived from stalled forks. Through epistasis experiments, we find that WNT, BMP and FGF have distinct roles during DNA replication, and demonstrate that WNT/GSK3 signalling sits at the helm of this regulatory cascade. Cell signalling control of chromosome segregation declines after pluripotency exit and specification into the three human germ layers, but re-emerges in differentiating neural progenitors. Through ex vivo and in vivo analyses, we also show that FGF and WNT signalling display opposite roles in chromosome segregation fidelity in mouse neural progenitors during the onset of neurogenesis (E14.5), but not during their expansion (E12.5). In particular, we find that the neurogenic factor FGF2 induces DNA replication stress-mediated chromosome missegregation, which could provide a rationale for the elevated chromosomal mosaicism of the developing brain. Our results highlight a role for patterning signals and cellular identity in genome maintenance that contributes to somatic mosaicism during mouse and human early lineage specification and neurogenesis. Overall design: hiPSC were treated with DMSO, Aphidicolin (200nM) or DKK1 (250ng/ml) for 3hrs prior to double pulse EdU labeling, single cells were sorted and subjected to scEdU-seq (van den Berg, et al., Nature Methods,2024

人类发育与内环境稳态依赖于遗传蓝图的正确复制、维持与分离。目前尚不明确这些细胞内过程如何在不同人类细胞谱系中受到监测调控，以及发育过程中镶嵌变异的时空分布差异背后的机制。本研究借助人类与小鼠多能干细胞（pluripotent stem cells），发现包括WNT、BMP与FGF在内的多种谱系特化信号，可汇聚调控S期内的DNA复制应激与损伤，进而控制有丝分裂中的纺锤体动态与染色体分离保真度。研究证实，哺乳动物原肠胚形成过程中与前向分化相关的模式化信号会加剧染色体错分离，而WNT与BMP这类后向分化信号则可保护多能干细胞免受过度的复制起始点激活、DNA损伤以及停滞复制叉引发的染色体错分离。通过上位性实验，本研究发现WNT、BMP与FGF在DNA复制过程中发挥各异的调控作用，并证实WNT/GSK3信号通路处于该调控级联的核心地位。多能性退出并分化为人类三胚层后，细胞信号对染色体分离的调控作用会显著减弱，但在分化的神经前体细胞中会重新激活。通过离体与体内分析，本研究还发现，在神经发生起始阶段（小鼠胚胎发育E14.5时期），FGF与WNT信号在小鼠神经前体细胞的染色体分离保真度中发挥相反调控作用，但在神经前体细胞扩增阶段（E12.5时期）并无此差异。具体而言，研究发现神经发生因子FGF2会诱导DNA复制应激介导的染色体错分离，这可为发育中大脑的高染色体镶嵌变异率提供合理的解释机制。本研究结果揭示了模式化信号与细胞身份在基因组维持中的关键作用，该作用可在小鼠与人类早期谱系特化及神经发生过程中促成体细胞镶嵌变异。整体实验设计：人类诱导多能干细胞（hiPSC, human induced pluripotent stem cells）在进行双脉冲EdU标记前，先用二甲基亚砜（DMSO）、阿非迪霉素（200nM）或DKK1（250ng/ml）处理3小时；随后分选单个细胞并开展scEdU-seq分析（引自van den Berg等人发表于《自然·方法学》（Nature Methods）2024年的研究）