Figure 1.

Generation of basal forebrain cholinergic neurons (BFCN) through sequential growth factor treatments. (A, B): Confocal microscopy demonstrates that cells generated through BMP9 treatment of fibroblast growth factor 8/sonic hedgehog pretreated neural progenitors express ChAT, p75, and Map2 and have a projection neuron morphology, whereas control neurons are only Map2-positive. (C, D): Confocal microscopy of equivalent cells stained only for ChAT and Map2 show the same BMP9 response and long ChAT+, Map2-axons. Scale bar = 20 μM. (E, F): BMP9-mediated ChAT immunopositivity is associated with expression of the VChaT. Lower magnification analysis of fields of neurons stained for VChAT. Scale bar = 100 μM. (G): qRT-PCR analysis shows 12- to 40-fold increases of RNA levels for markers characteristic of the BFCN. Bars are standard error, n = 4. All increases were significant by ANOVA (*, p < .0001; **, p = .0014; ***, p < .0001; ****, p < .0001). Data are from four replicate experiments; error bars show SEM. Data in (A–F) are from five replicate experiments, data in (G) is from four replicate experiments; error bars show SEM. Abbreviations: AChE, acetylcholinesterase; BMP9, bone morphogenetic protein-9; ChAT, choline acetyltransferase; Map2, microtubule-associated protein 2; VChAT, vesicular acetylcholine transporter.