Time-Course Gene Expression Profiling Reveals a Novel Role of Non-Canonical WNT Signaling During Neural Induction

The process of neuroepithelial differentiation from human pluripotent stem cells (PSCs) resembles in vivo neuroectoderm induction in the temporal course, morphogenesis, and biochemical changes. This in vitro model is therefore well-suited to reveal previously unknown molecular mechanisms underlying neural induction in humans. By transcriptome analysis of cells along PSC differentiation to early neuroepithelia at day 6 and definitive neuroepithelia at day 10, we found downregulation of genes that are associated with TGF-β and canonical WNT/β-CATENIN signaling, confirming the roles of classical signaling in human neural induction. Interestingly, WNT/Ca(2+) signaling was upregulated. Pharmacological inhibition of the downstream effector of WNT/Ca(2+) pathway, Ca(2+)/calmodulin-dependent protein kinase II (CaMKII), led to an inhibition of the neural marker PAX6 and upregulation of epidermal marker K18, suggesting that Ca(2+)/CaMKII signaling promotes neural induction by preventing the alternative epidermal fate. In addition, our analyses revealed known and novel expression patterns of genes that are involved in DNA methylation, histone modification, as well as epithelial-mesenchymal transition, highlighting potential roles of those genes and signaling pathways during neural differentiation.