Limb reduction in an Esco2 cohesinopathy mouse model is mediated by p53-dependent apoptosis and vascular disruption [scRNA-Seq]

Roberts syndrome (RBS) is an autosomal recessive disorder with profound growth deficiency and limb reduction caused by ESCO2 loss-of-function mutations. We elucidate the pathogenesis of limb reduction in an Esco2fl/fl;Prrx1-CreTg/0 mouse model using bulk- and single cell-RNA-seq and gene co-expression network analyses during embryogenesis. Our results reveal morphological and vascular defects culminating in hemorrhage of mutant limbs at E12.5. Underlying this abnormal developmental progression is a pre-apoptotic, mesenchymal cell population specific to mutant limb buds enriched for p53-related signaling beginning at E9.5. We then characterize these p53-related processes of cell cycle arrest, DNA damage, cell death, and the inflammatory leukotriene signaling pathway in vivo. In utero treatment with the p53 inhibitor pifithrin-, identified using a drug repositioning approach, rescued the hemorrhage in mutant limbs. Lastly, significant enrichments were identified among genes associated with RBS, thalidomide embryopathy, and human limb reduction disorders, suggesting etiological commonalities of cell death and vascular defects. Libraries were generated based on three replicates of each genotype (Esco2fl/fl;Prrx1-CreTg/0 and Esco2fl/fl). Each replicate consisted of 3 pooled samples (2 limb buds per sample, comprising a total of 6 limb buds per replicate). Samples were genotyped by PCR and each replicate was sex balanced with two male samples and one female sample.罗伯茨综合征（Roberts syndrome, RBS）是一类由ESCO2功能丧失性突变引发的常染色体隐性遗传病，以严重生长发育缺陷与肢体发育不全为主要特征。本研究通过胚胎发生阶段的批量及单细胞RNA测序（bulk- and single cell RNA-seq）与基因共表达网络分析，在Esco2fl/fl;Prrx1-CreTg/0小鼠模型中阐明了肢体发育不全的发病机制。研究结果显示，突变体肢体在胚胎发育第12.5天（E12.5）出现形态学与血管异常，最终引发肢体出血。该异常发育进程的细胞学基础为：自胚胎发育第9.5天（E9.5）起，突变肢体芽中出现特异性的前凋亡间充质细胞群，该细胞群显著富集p53相关信号通路。本研究进一步在体内表征了该p53相关的细胞周期阻滞、DNA损伤、细胞死亡及炎症性白三烯信号通路过程。通过药物重定位策略筛选得到的p53抑制剂Pifithrin-可通过宫内给药挽救突变体肢体的出血表型。最后，本研究发现与RBS、沙利度胺胚胎病及人类肢体发育不全疾病相关的基因存在显著富集，提示三者在细胞死亡与血管缺陷方面存在共同的病因学共性。本研究的测序文库基于两种基因型（Esco2fl/fl;Prrx1-CreTg/0与Esco2fl/fl）各3次生物学重复构建：每次重复包含3份混合样本，每份样本含2个肢体芽，即每个重复总计6个肢体芽。所有样本均通过PCR进行基因型鉴定，且每次重复均实现性别平衡，包含2份雄性样本与1份雌性样本。