To generate very homogeneous populations of ventral telencephalic neuronal subtypes, we have used temporal and combined modulation of developmentally relevant signaling pathways. Specifically, we have employed (i) induction of neuroectoderm formation by dual SMAD inhibition [26], (ii) inhibition of Wnt signaling, which otherwise caudalizes [33,34] and dorsalizes [35] differentiating neuroectoderms, (iii) strong activation of SHH signaling, which ventralizes neuroectoderms [2], and (iv) activation of FGF8 or FGF19 signaling, which induces the MGE or CGE phenotype, respectively, at the expense of the other phenotype. According to our previous study, a more efficient ventral telencephalic phenotype induction is achieved by early modulation of Wnt and SHH signaling pathways, even before neuroectoderm formation is completed [21]. This is consistent with previous developmental studies, which have shown that early inhibition of Wnt signaling is important for telencephalic induction of the neural plate [33,34], and early SHH signaling in the anterior neural plate at the gastrula stage induces the prospective ventral telencephalon, [36] even prior to neural tube formation.
