Fig. S6

TLX and OC activity control neural stem cells fate (related to Figure 4)

(A) In vitro effect of transfecting primary human embryonic neural stem cells with a control EGFP plasmid, TLXp2A- EGFP or OC2-p2A-EGFP. Overexpression of TLX (n=646) and OC2 (n=528) significantly depletes the existing neural stem cell population in the dish compared to the EGFP control (n=408). Furthermore, in comparison with EGFP transfection (n=344), expression of TLX (n=99) and OC2 (n=69) slightly increases the percentage of primary human NSCs that differentiate into cortical neurons. (B) Triple immunolabelling against TUJ1, EGFP and VEGF-A after transfection of primary human embryonic NSCs with the TLX-p2A-EGFP plasmid (nuclear marker DAPI is in blue). Embryonic cortical neuron express high detectable level of nuclear (arrowhead) and cytoplasmic (arrow) VEGF-A after TLX expression (EGFP). (C) Confocal projection of immunolabelling with endogenous GS protein in the brain of control MO or miR-9 MO injected larvae and larvae expressing uas:tlx or uas:ocl in NSCs using Tg(gfap:gal4) line at 72 hpf. Embryonic NSCs are reduced in the miR-9 depleted and tlx or ocl expressing brain. (D) Whole-mount in situ hybridization against deltaA, ascl1a, ascl1b, neurog1 or huc in embryos at 48 hpf injected with the control MO, miR-9 MO, tlx TP or ocl TP. miR-9 inhibition increases neural progenitor cells (deltaA and ascl1a) and promote a neuronal fate (huc). tlx or ocl mRNA protection also leads to an increase in deltaA+ neural progenitors in the zebrafish brain. Of note, miR-9 morphant shows an increase in deltaA+ and ascl1a+ NPCs but not ascl1b+ or neurog1+ NPCs. Dorsal view, Anterior up. Scale bars: 10 μm (B) or 100 μm (C, D). Error bars represent s.d. *P<0.05, **P<0.001, ***P<0.0005, determined by t-test, two-tailed.