FIG. 7.

Overexpression of Ngns and Mash1 promotes neuronal subtype specification. (A) Immunofluorescent labeling against the neuronal subtype-specific markers GAD67 (red, first line), GABA (green, second line), HB9 (green, third line), and ChAT (green, fourth line) in hNPC populations transduced with the VSV-PGK-LacZ, VSV-PGK-Ngn1, VSV-PGK-Ngn2, VSV-PGK-Ngn3, and VSV-PGK-Mash1 vectors. Control hNPCs (LacZ) spontaneously gave rise to GAD67⁺/GABA⁺ GABAergic, ChAT⁺ cholinergic, and HB9⁺ motor neurons. Overexpression of Ngn1 did not promote hNPC differentiation into any of these lineages. On the contrary, overexpression of Ngn2 strongly increased the expression of GAD67, HB9, and ChAT and, to a lesser extent, of GABA. Ngn3 strongly increased the expression of GAD67 and HB9 and, to a lesser extent, of GABA and ChAT. Finally, Mash1 increased the expression of both GAD67 and GABA, as well as HB9, but displayed its strongest effect on the expression of ChAT. Scale bar: 200 μm. (B) Semiquantitative evaluation of the effects of Ngn1, Ngn2, Ngn3, and Mash1 overexpression on terminal neuronal differentiation of hNPCs, compared with control cells transduced with the VSV-PGK-LacZ vector. (+/−) <1% positive cells; (+) 1%–20% positive cells; (++) 20%–50% positive cell; (↔) no effect; (↓↔) <10% positive cell decrease; (↔↑) <10% positive cell increase; (↑) 10%–50% positive cell increase; (↑↑) >50% positive cell increase.