Limb reduction in an Esco2 cohesinopathy mouse model is mediated by p53-dependent apoptosis and vascular disruption [RNA-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. A total of 18 libraries were generated based on triplicate samples of each genotype at each stage (three replicates of Esco2fl/fl;Prrx1-CreTg/0 and Esco2fl/fl at E9.5, E10.5 and E11.5). Libraries were then sequenced on the Illumina NovaSeq 6000 (300 cycle paired-end sequencing with a read length of 150 bp). For each genotype at each age, pairwise comparisons were performed to identify differentially expressed genes between mutant and control limb buds.罗伯茨综合征（Roberts syndrome, RBS）是一类由ESCO2功能丧失突变引发的常染色体隐性遗传病，核心表现为严重生长发育缺陷与肢体发育不全。本研究以Esco2fl/fl;Prrx1-CreTg/0小鼠模型为研究对象，采用体转录组测序（bulk RNA-seq）、单细胞RNA测序（single cell RNA-seq, scRNA-seq）联合基因共表达网络分析的策略，阐明了胚胎发育过程中肢体发育不全的致病机制。研究结果显示，突变体肢体在胚胎发育第12.5天（E12.5）出现形态学与血管缺陷，最终引发出血。该异常发育进程的细胞学基础为：自胚胎第9.5天（E9.5）起，突变肢体芽中出现特异性的凋亡前间质细胞群，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、细胞死亡以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p53抑制剂pifithrin-，对孕鼠实施宫内给药后，成功挽救了突变体肢体的出血表型。最后，本研究鉴定发现，与RBS、沙利度胺胚胎病以及人类肢体发育不全疾病相关的基因存在显著富集，提示细胞死亡与血管缺陷存在共同的致病机制。本研究共构建18个转录组测序文库，样本基于每个发育阶段、每种基因型的三份生物学重复获得：即E9.5、E10.5和E11.5三个发育阶段的Esco2fl/fl;Prrx1-CreTg/0突变体与Esco2fl/fl对照组，各设置三次生物学重复。随后使用Illumina NovaSeq 6000测序平台进行300循环双端测序，读长为150 bp。针对每个发育阶段的每种基因型，均开展组间两两比较，以鉴定突变体与对照组肢体芽之间的差异表达基因。