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Retinoic acid and Wnt/β‐catenin signals play important roles during neuronal differentiation but less is known about noncanonical Wnt signals in this context. We examined retinoic acid and Wnt signaling in two human embryonal carcinoma cell lines, NTERA‐2 (clone D1), which undergoes neuronal differentiation in response to retinoic acid, and 2102Ep, which does not. Retinoic acid treatment inhibited β‐catenin/Tcf activity in NTERA‐2 cells but not in 2102Ep cells. Inhibition occurred downstream of β‐catenin but did not involve competition between retinoic acid receptors and β‐catenin for binding to p300 or Tcf‐4. Ectopic expression of FZD1 partially restored inhibition in 2102Ep cells, suggesting the involvement of Wnt ligands. Retinoic acid treatment of NTERA‐2 cells induced the expression of Wnt‐4 and Wnt‐11, both of which were able to inhibit β‐catenin/Tcf activity. Wnt‐4 and Wnt‐11 were found at cell borders in islands of cells that expressed OCT4 and GFAP and were predominantly negative for Nestin, PAX6, and GATA6. Gene silencing of Wnt‐4, but not Wnt‐11, reduced retinoic acid downregulation of OCT4 and Nanog and upregulation of PAX6, ASCL1, HOXC5, and NEUROD1, suggesting that Wnt‐4 promotes early neuronal differentiation. Gene expression analysis of NTERA‐2 cells stably overexpressing Wnt‐11 suggested that Wnt‐11 potentiates retinoic acid induction of early neurogenesis. Consistent with this, overexpression of Wnt‐11 maintained a population of proliferating progenitor cells in cultures treated with retinoic acid for several weeks. These observations highlight the distinct roles of two noncanonical Wnts during the early stages of retinoic acid‐induced neuronal differentiation. S TEM  C ELLS  2011;29:141–153

Distinct Roles for Wnt‐4 and Wnt‐11 During Retinoic Acid‐Induced Neuronal Differentiation