SOX21 ensures rostral forebrain identity by suppression of WNT8B during neural regionalization of human embryonic stem cells

Generation of brain region-specific progenitors from human embryonic stem cells (hESCs) is critical for their application. However, transcriptional regulation of neural regionalization in human embryos is poorly understood. Here, we report that transcription factor SOX21 is required for telencephalic fate specification from hESCs. SOX21 knockout (KO) impairs telencephalic fate specification, concomitant with the activation of diencephalic genes and Wnt signaling. Inhibition of Wnt signaling restores telencephalic specification from SOX21 KO hESCs. Analysis of global SOX21 DNA binding profiles and SOX21-regulated genes identifies SOX21 targets. SOX21 interacts with b-catenin and interferes with TCF4/b-catenin binding to the enhancer of WNT8B. Double KO of SOX21 and WNT8B restores telencephalic specification, indicating that SOX21 specifies the telencephalic fate through repressing WNT8B expression. Moreover, the high and low levels of SOX21 in the telencephalon and diencephalon, respectively, in developing human brains support its role in telencephalic fate specification. Collectively, this study unveils previously unappreciated roles of SOX21 and sheds light on the transcriptional control of telencephalic patterning in humans. Overall design: To dissect the transcriptional network controlling the rostrocaudal specification of early neural differentiation, we generated NPCs from hESCs with defined regional identities ranging from the rostral forebrain to the spinal cord based on published protocols (Imaizumi et al., 2015; Kirkeby et al., 2012; Kriks et al., 2011). Generally, rostrocaudal patterning was established by the dose-dependent activation of WNT signaling. Inhibition of Wnt signaling by IWP2 (2 uM) induced the rostral forebrain fate, while activation of Wnt signaling by CT induced the diencephalic, mesencephalic and rhombencephalic fate, depending on CT concentrations ranging from 0.4 to 4.0 uM. RA (1.0 uM) coupled with 4.0 uM CT (CT4.0RA) was used to induce the spinal cord fate. We then carried out RNA sequencing (RNA-seq) for cell samples collected from 19 groups (each with two biological replicates), covering 3 time points (days 0, 4, 8) and 9 differentiation conditions (IWP2, CT0.0, CT0.4, CT0.8, CT1.0, CT2.0, CT3.0, CT4.0, CT4.0RA).