Antagonism between the transcription factors NANOG and OTX2 specifies rostral or caudal cell fate during neural patterning transition

Abstract

During neurogenesis, neural patterning is a critical step during which neural progenitor cells (NPCs) differentiate into neurons with distinct functions. However, the molecular determinants that regulate neural patterning remain poorly understood. Here, we optimized the "dual-SMAD inhibition" method to specifically promote differentiation of human pluripotent stem cells (hPSCs) into forebrain and hindbrain NPCs along the rostral-caudal (R-C) axes. We report that neural patterning determination occurs at the very early stage in this differentiation. Undifferentiated hPSCs expressed basal levels of the transcription factor orthodenticle homeobox 2 (OTX2) that dominantly drove hPSCs into the "default" rostral fate at the beginning of differentiation. Inhibition of glycogen synthase kinase 3β (GSK3β) through CHIR99021 (CHIR) application sustained transient expression of the transcription factor NANOG at early differentiation stages through Wnt signaling. Wnt signaling and NANOG antagonized OTX2 and in the later stages of differentiation, switched the default rostral cell fate to the caudal one. Our findings have uncovered a mutual antagonism between NANOG and OTX2 underlying cell fate decisions during neural patterning, critical for the regulation of early neural development in humans.The embryonic neurodevelopment is a spatiotemporally regulated process, during which http://www.jbc.org/ Downloaded from 2 distinct cell fates are progressively restricted based on spatial regions (1, 2). At early stage of neurogenesis, the specified neural ectoderm divides into functionally distinct cell fates along the anterior-posterior (A-P) and dorsal-ventral (D-V) axes (3). This neural patterning process is a critical step to specify different neural precursors such as forebrain, midbrain, hindbrain and spinal cord. It has been known that the neural patterning is induced by the temporal and special morphogen gradients along the A-P and D-V axes (4, 5). These morphogens, including BMPs, WNTs, FGFs, RA and SHH (sonic hedgehog), coordinate and form gradients to specify regionally transcriptional program and distinct neural progenitors (6-10). However, the precise timing and mechanisms underlying morphogeninduced neuraxial patterning has not been fully elucidated in mammals, especially in human.Human pluripotent stem cells (hPSCs), including embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSC) could differentiate into neuroepithelial cells , regionally specified neural precursor cells (11-16), thus provides a valuable model to investigate molecular determinants on neural patterning in human background. The mostly used method to induce neural differentiation in hPSCs is through suppression of both TGFβ and BMP signaling (17,18). Dual-inhibition of SMAD-dependent TGF-β and BMP signaling by their inhibitors (SB431542 and Noggin), or SB431542 and Dorsomorphin can efficiently trigger hPSCs differentiation into NPCs (19-21). Dual-SMAD inhibition triggered NPCs are believed to be more close to the anterior forebrai...

JMJD3 and UTX determine fidelity and lineage specification of human neural progenitor cells

“…1c-i), indicating that JMJD3 and UTX are dispensable in maintaining hESCs. We then triggered default neural differentiation in these cells under defined conditions through the dual inhibition of TGFb/BMP signalling 3,41 (Fig. 1a).…”

“…4h). All the top ten TF motifs most enriched in dKO-closed regions have been reported to be essential neural regulators, for example, RFX2 (P = 1e −1064 ), SOX2 (P = 1e −661 ), and OTX2 (P = 1e −403 ) 41,44,45 (Fig. 4h).…”

“…Wild-type hES cells and three KDM6 mutant hES cell lines lacking UTX, JMJD3 or both at 95-100% cell confluence were seeded onto Matrigel-coated 12-well plates in mTeSR1 medium. These cells were cultured in N2B27 medium (50% DMEM/F12 (Gibco), 50% neurobasal (Gibco), 0.5 × N2 (Gibco), 0.5 × B27 (Gibco), 1% Glutamax (Gibco), 1% NEAA (Gibco), 5 μg mL −1 insulin (Gibco), and 1 μg mL −1 heparin (Sigma)) plus 5 μM SB431542 (Selleck) and 5 μM dorsomorphin (Selleck) 41 . The medium was changed every two days.…”

“…Neural progenitor cells (NPCs) undergo random differentiation. Wild-type NPCs and three KDM6 mutant NPCs lacking UTX, JMJD3 or both (NPC-P2) at passage two were digested into single cells and used for random differentiation 41 . NPCs (2 × 10 4 ) were seeded onto Matrigel-coated 24-well plates and cultured in NPC medium plus 10 μM Y-27632.…”

Derivation of Specific Neural Populations From Pluripotent Cells for Understanding and Treatment of Spinal Cord Injury

“…Dorsalization of neuronal subtypes, as mentioned previously, is driven through WNT and BMP signaling, and the single publication (to our knowledge) on producing dorsal INs from stem cells relied on recombinant proteins (Gupta et al, 2018). Although it should be noted that plenty of small molecule BMP/TGFβ activators and inhibitors exist, and similar to the previously mentioned SMAD inhibitors used for hPSC induction (Chambers et al, 2009;Su et al, 2018).…”