One surprising finding from the gene expression data was the induction of FOXA2 in all SHH treated cultures (Figure 2I). Expression of FOXA2, a marker thought to be largely restricted to the floor plate within the developing CNS, was confirmed at day 18 of differentiation in NKX2.1::GFP+ cells of all three SHH treatment paradigms (Figure S1A,B). To address whether FOXA2 expression represents an in vitro artifact following strong extrinsic activation of SHH signaling, or whether FOXA2 expression occurs in the telencephalon during in vivo development, we performed histological analyses in the developing mouse and human forebrain. In mouse embryos (E11.5) FOXA2 expression within the mouse prosencephalon was segregated from the FOXG1 and NKX2.1 domains (Figure S1C). However, immunohistochemical analysis for FOXA2 in primary human forebrain tissue (CS15 embryo) confirmed expression in the telencephalic NKX2.1+ ganglionic eminence (Figure S1D–F). These in vivo data are in agreement with the in vitro hPSC differentiation data demonstrating (transient) FOXA2 expression during human ventral forebrain development.
