Single-cell RNA-seq identifies a reversible mesodermal activation in abnormally specified epithelia of p63 EEC syndrome

Abstract

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.

TP63 links chromatin remodeling and enhancer reprogramming to epidermal differentiation and squamous cell carcinoma development

“…This suggests that TP63 or ΔNp63 is dispensable for epidermal lineage commitment. Intriguingly, introducing p63 mutants from EEC patients into induced pluripotent stem cells (iPSCs) dramatically impairs the induction of epidermal marker K14 in an epidermal commitment model [ 58 ] induced by retinoic acid (RA) and bone morphogenetic protein 4 (BMP4) [ 59 ], arguing that TP63 is essential for epidermal commitment. These opposite interpretations may indicate that other lineage-determining factors could compensate for p63 function in a p63-null context, whereas mutant p63 in iPSCs abrogates the function of the normal p63.…”

“…It has been shown that genes expressed at an early differentiation stage are not under the control of p63. The p63 protein predominantly regulates genes during the specification switch from the multipotent state to the epidermal fate [ 59 ]. Thus, these studies suggest that p63 plays a prominent role in maturation, rather than the initiation stage of skin differentiation triggered by inductive morphogens (Table 1 ).…”

“…EEC syndrome patients display keratinocytes harboring p63 mutations, which lack DNA-binding ability, and display a loss of enhancers at epidermal-expressing genes, indicating that ΔNp63α binding of DNA is necessary to establish the epidermal enhancer landscape and epidermal commitment [ 64 , 77 ]. Furthermore, the majority of p63-bound epidermal enhancers lose their accessibility in p63 knockout ESC during in vitro differentiation [ 59 ]. Taken together, these studies indicate that the formation and maintenance of epidermal enhancers at p63-binding sites are heavily dependent on p63 expression and its binding to DNA (Table 1 ).…”

“…Introducing p63 mutants from EEC patients into iPSCs impairs the acquisition of epithelial identity, but also activates mesodermal genes expression. In contrast, inhibition of mesodermal induction by suramin enhances epidermal differentiation of iPSCs carrying the p63 mutation [ 59 ]. Cbx4, a component of polycomb repressive complex 1 (PRC1), is a target of p63 and contributes to repression of nonepithelial genes during epidermal differentiation [ 79 ].…”

Lyophilized powder of mesenchymal stem cell supernatant attenuates acute lung injury through the IL-6–p-STAT3–p63–JAG2 pathway

“…Data from numerous studies have demonstrated that p63, a homolog of p53, is vital for the normal development and homeostasis of epithelial tissues, in both humans and mice. For instance, heterozygous mutations in human p63 drives several developmental defects and disorders, especially skin abnormalities (47)(48)(49). In animal studies, p63 knockout led to severe anomalies in the development of epithelia and their derivatives, and even death at birth (25,50).…”