Fig. 7. Shh regulates proliferation and neurosphere formation in cooperation with EGF. (A) Image of neurospheres from adult SVZ cultures. (B) Quantification of the number of primary neurospheres formed from cultures of SVZ cells previously grown on astrocytic monolayers with or without exogenous Shh (5 nM). Shh was not added to the neurosphere cultures. (C) Synergism of Shh and EGF on neurosphere proliferation. The assay was done with a constant dose of EGF at 1 ng/ml, and varying doses of Shh at 5 or 0.5 nM (left) or with a constant does of Shh at 5 nM and varying doses of EGF at 5 and 0.5 ng/ml (right). Treatments were for 48 hours. (D,E) Quantification of proliferation as measured by the percentage of BrdU⁺ cells (D) and the number of clones obtained in cloning assays (E) in adult SVZ neurospheres treated with cyclopamine (5 µM) or treated with an equal dose of ethanol used as carrier for in vitro work. In all cases, error bars indicate s.e.m. of triplicate cultures.