The impact of timing of SHH exposure on the derivation of specific ventral precursor populations was further examined by global transcriptome analysis (Figure 2I–K, Table S1, all raw data are available on GEO: http://www.ncbi.nlm.nih.gov/geo/: Accession # pending). A direct comparison of day 10-18 versus untreated cultures (No SHH) confirmed the robust induction of ventral specification markers such as NKX2.1, NKX2.2, ASCL1, SIX6, OLIG2, and NKX6.2, which were induced at the expense of dorsal forebrain markers such as EMX2 and PAX6 (Figure 2I). There were no significant differences in the expression of general anterior markers such as FOXG1 and OTX2 (Figure 2I), supporting the notion that both populations are of forebrain identity. Comparison of day 10-18 to day 2-18 treated cultures (Figure 2J) illustrated differences in the expression of anterior markers such as FOXG1 versus diencephalic markers such as RAX. In contrast, day 10-18 versus day 6-18 treated cultures showed less pronounced differences in forebrain marker expression (Figure 2K). Overall, our data demonstrate that the window of SHH treatment significantly impacts identity of hPSC-derived NKX2.1 precursors.
