Single-cell RNA-seq identifies a reversible epithelial-mesenchymal transition in abnormally specified epithelia of p63 EEC syndrome

Mutations in transcription factor p63 are associated with developmental disorders that manifest defects in stratified epithelia including the epidermis. The underlying cellular and molecular mechanism is however not yet understood. We established an epidermal commitment model using human induced pluripotent stem cells (iPSCs) and characterized differentiation defects of iPSCs derived from ectrodactyly, ectodermal dysplasia, and cleft lip/palate (EEC) syndrome patients carrying p63 mutations. Transcriptome analyses revealed distinct step-wise cell fate transitions during epidermal commitment; from multipotent simple epithelium to basal stratified epithelia, and ultimately to the mature epidermal fate. Differentiation defects of EEC iPSCs caused by mutant p63 occurred during the specification switch from the simple epithelium to the basal stratified epithelial fate. Single-cell transcriptome and pseudotime analyses identified signatures of embryonic epithelial-mesenchymal transition (EMT) associated with the deviated commitment route of EEC iPSCs. Repressing mesodermal activation reversed the EMT and enhanced epidermal commitment. Our findings demonstrate that p63 is required for specification of stratified epithelia, probably by repressing embryonic EMT during epidermal commitment. This study provides insights into disease mechanisms underlying stratified epithelial defects caused by p63 mutations and suggests potential therapeutic strategies for the disease.\n\nSignificance statementMutations in p63 cause several developmental disorders with defects of epithelial related organs and tissues including the epidermis. Our study is to dissect the unknown cellular and molecular pathomechanism. We utilized human induced pluripotent stem cells (iPSCs) derived from ectrodactyly, ectodermal dysplasia, and cleft lip/palate (EEC) syndrome patients carrying p63 mutations and studied transcriptome changes during differentiation of these cells to epidermal cells. Our analyses showed that the specification of the proper epithelial cell fate was affected by p63 EEC mutations, with an abnormal embryonic epithelial-mesenchymal transition (EMT). Repressing mesodermal activation reversed the EMT and enhanced epidermal commitment. This study provides insights into disease mechanisms associated with p63 mutations and suggests potential therapeutic strategies.