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-a, 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. Overall design: 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.