Figure 7.

Genetic suppression of GLI3 extends the window of competency toward MN fate. A, qRT-PCR confirms suppression of GLI3 mRNA during neural induction in DOX-treated RFP⁺ cells but not RFP⁻ cells or untreated cells using our DOX-inducible shRNAmir GLI3-RFP hESC line. Data are shown as mean ± SEM; n = 3 independent experiments; **p < 0.01; *p < 0.05 versus LSB, Dunnett test. B, Diagram depicting patterning paradigm for experiment. C, Quantification of immunocytochemistry at day 21 for HB9 and ISL1 shows that GLI3 suppression via DOX treatment significantly rescues ability of RA to derive MNs from hPSCs at a later neural stage. Data are shown as mean ± SEM; n = 3 independent experiments; ****p < 0.0001; ***p < 0.001; **p < 0.01 versus RA, Dunnett test; late patterning compared with late patterning + DOX using unpaired t tests, two-tailed. D, Immunocytochemistry at day 23 for ISL1 (red), HB9 (green), and TUJ1 (blue) demonstrates the ability to specify MNs with similar efficiency to early patterning when later patterning stages are combined with DOX-induced GLI3 suppression. Scale bars, 100 μm. E, PCR amplification of GLI3 from genomic DNA of control (H9 and GLI3.3) and knock-out lines (GLI3.1 and GLI3.2) confirms a migrational shift of the PCR products from the knock-out lines consistent with genomic deletion. F, Western blot for GLI3 verifies loss of GLI3 protein in CRISPR GLI3 knock-out hESC lines (GLI3.1 and GLI3.2) but not the untargeted control line (GLI3.3). G, Immunocytochemistry at day 29 for HB9 (red), ISL1 (green), and TUJ1 (blue) shows knock-out but not control lines have the ability to specify MNs at later patterning stages with similar efficiency to early patterning of control line. H, Quantification of immunocytochemistry at day 21 for ISL1, HB9, and NKX6.1 shows that GLI3 knock-out rescues the ability of RA to derive MNs from hPSCs at a later neural stage. Data are shown as mean ± SEM; n = 3 independent experiments; ****p < 0.0001; **p < 0.01 versus late RA in GLI3.3 line, Dunnett test.