Fig. 7. FGF and Wnt induce phase transition to generate the CM.

(A) Deletion of either β-catenin alone (βcat^ΔRet) or β-catenin and Fgfrs together (Fgfr^ΔRet;βcat^ΔRet) transformed the distal retina into the NR as indicated by loss of Cdo and Msx1, lack of Otx2 and Pcad, and expansion of Sox2 (all at 100% in the β-catenin or β-catenin/pERK–deficient regions marked by dotted lines; n = 3). (B) Although overexpression of Wnt1 in Wnt1^OE embryos did not produce any phenotype in the retina, when combined with overexpression of Fgf8, it transformed the RPE domain of the Wnt1^OE;Fgf8^OE eye cup into the CM as indicated by the up-regulation of the CM-specific genes Otx1 and Cdo and the lack of the RPE marker Mitf and the NR marker Atoh7. (C) Untreated hiPSC organoid culture contained few MSX1⁺ or CDO⁺ CM-like cells. Addition of the 3 μM WNT agonist CHIR99021 significantly expanded the number of MSX1⁺ and CDO⁺ cells. (D) Quantification of the MSX1⁺ and CDO⁺ area as the percentage of the total organoid culture. (E) FGF and Wnt signaling promotes the abrupt and reversible transition of eye cup progenitors into three phases: the NR, the CM, and the RPE. Our study showed that the NR can be transformed by constitutive activation of Wnt signaling (βcat^CA) into the CM, which is converted to the RPE after the loss of FGF signaling (Fgfr^Δ). The RPE reverts back to the CM by titrating FGF signaling (Fgf8^OE;Fgfr^Δ). Otherwise, ablation of Wnt signaling (βcat^Δ) or overexpression of Fgf8 (Fgf8^OE) can turn the RPE into the NR, which may transition further into the CM after overexpression of Wnt1 (Wnt1^OE). Scale bars, 100 μm.