Department of Molecular Developmental Biology, Faculty of Science, Radboud Institute for Molecular Life Sciences, Radboud University, 6525GA Nijmegen, The Netherlands.
Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) Foundation, Ministry of Education of Brazil, 70.040-031 Brasília, Brazil.
Proc Natl Acad Sci U S A. 2019 Aug 27;116(35):17361-17370. doi: 10.1073/pnas.1908180116. Epub 2019 Aug 14.
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 stepwise cell fate transitions during epidermal commitment: Specification from multipotent simple epithelium to basal stratified epithelia and ultimately to the mature epidermal fate. Differentiation defects of EEC iPSCs caused by p63 mutations occurred during the specification switch from the simple epithelium to the basal-stratified epithelial fate. Single-cell transcriptome and pseudotime analyses of cell states identified mesodermal activation that was associated with the deviated commitment route of EEC iPSCs. Integrated analyses of differentially regulated genes and p63-dependent dynamic genomic enhancers during epidermal commitment suggest that p63 directly controls epidermal gene activation at the specification switch and has an indirect effect on mesodermal gene repression. Importantly, inhibitors of mesodermal induction enhanced epidermal commitment of EEC iPSCs. Our findings demonstrate that p63 is required for specification of stratified epithelia, and that epidermal commitment defects caused by p63 mutations can be reversed by repressing mesodermal induction. This study provides insights into disease mechanisms underlying stratified epithelial defects caused by p63 mutations and suggests potential therapeutic strategies for the disease.
转录因子 p63 的突变与发育障碍有关，这些障碍表现为包括表皮在内的分层上皮缺陷。然而，其潜在的细胞和分子机制尚不清楚。我们使用人类诱导多能干细胞（iPSC）建立了表皮分化模型，并对携带 p63 突变的并指畸形、外胚层发育不良和唇腭裂（EEC）综合征患者的 iPSC 分化缺陷进行了特征分析。转录组分析显示，在表皮分化过程中存在逐步的细胞命运转变：从多能简单上皮细胞向基底分层上皮细胞，最终向成熟表皮细胞分化。由 p63 突变引起的 EEC iPSC 分化缺陷发生在简单上皮细胞向基底分层上皮细胞命运分化的转换过程中。单细胞转录组和细胞状态的拟时分析确定了中胚层的激活，这与 EEC iPSC 偏离的分化途径有关。在表皮分化过程中差异调控基因和 p63 依赖的动态基因组增强子的综合分析表明，p63 直接控制着表皮基因在分化转换中的激活，并对中胚层基因的抑制有间接影响。重要的是，中胚层诱导抑制剂增强了 EEC iPSC 的表皮分化。我们的研究结果表明，p63 是分层上皮细胞分化所必需的，并且由 p63 突变引起的表皮分化缺陷可以通过抑制中胚层诱导来逆转。这项研究为 p63 突变引起的分层上皮缺陷的疾病机制提供了新的见解，并为该疾病的潜在治疗策略提供了思路。
