Wnt signal activation induces midbrain specification through direct binding of the beta-catenin/TCF4 complex to the EN1 promoter in human pluripotent stem cells

The canonical Wnt signal pathway plays a pivotal role in anteroposterior patterning and midbrain specification during early neurogenesis. Activating Wnt signal has been a strategy for differentiating human pluripotent stem cells (PSCs) into midbrain dopaminergic (DA) neurons; however, the underlying molecular mechanism(s) of how the Wnt signal drives posterior fate remained unclear. In this study, we found that activating the canonical Wnt signal significantly upregulated the expression of EN1, a midbrain-specific marker, in a fibroblast growth factor signal-dependent manner in human PSC-derived neural precursor cells (NPCs). The EN1 promoter region contains a putative TCF4-binding site that directly interacts with the β-catenin/TCF complex upon Wnt signal activation. Once differentiated, NPCs treated with a Wnt signal agonist gave rise to functional midbrain neurons including glutamatergic, GABAergic, and DA neurons. Our results provide a potential molecular mechanism that underlies midbrain specification of human PSC-derived NPCs by Wnt activation, as well as a differentiation paradigm for generating human midbrain neurons that may serve as a cellular platform for studying the ontogenesis of midbrain neurons and neurological diseases relevant to the midbrain. Brain development: Specifying differentiation into midbrain cells An evolutionarily conserved signaling pathway triggers the differentiation of human pluripotent stem cells (hPSCs) into functional midbrain neurons. Dong-Wook Kim at Yonsei University, South Korea, and colleagues explored the mechanisms through which the Wnt signal regulates neuronal cell fate. They found that both Wnt and fibroblast growth factor signaling are required to increase the expression of EN1 , a midbrain-specific gene, in a neural precursor cell population derived from hPSCs. They showed that activation of the Wnt signaling pathway leads to the formation of a protein complex containing beta-catenin that directly interacts with the promoter region of this gene to initiate transcription. Insights into how stem cells differentiate into midbrain-specific cell types will aid our understanding of neurological disorders affecting this brain region, such as Parkinson’s disease, and may lead to identification of novel therapeutic targets.