Fig. 6. Shh signaling regulates SVZ proliferation and neurogenesis. (A) Quantification of the effects of Shh on the proliferation of dissociated P5 SVZ cells plated on a quiescent astrocytic monolayer. BrdU incorporation was quantified by immunofluorescence. Under these conditions, SVZ precursors proliferate and generate new neurons, as they normally do in vivo. (B) Quantification of the effects of blocking anti-Shh monoclonal antibody (5E1) on the proliferation of P5 SVZ cells after dissociation and reaggregation. Cell proliferation was measured by radioactive thymidine incorporation. (C) Quantification of the effect of Shh on neurogenesis in dissociated adult SVZ cells plated on an astrocytic monolayer. Generation of new neurons was measured by co-labeling with Tuj1, identifying neurons, and anti-BrdU antibodies, identifying cells that replicated after BrdU addition. (D) Quantification of the effects of Shh on isolated type A SVZ neuroblasts. Type A cells were purified from P5 mice and cultured with or without Shh. At 3 and 7 days, the number of Tuj1+ cells in Shh-treated cultures were compared to control cultures. Error bars=s.e.m. (E) Immunocytochemistry of a 7-day SVZ cell culture on an astrocytic monolayer showing the labeling of neurons with Tuj1 (red) and recently divided cells with anti-BrdU (green) antibodies. Note the large number of doubly labeled (yellow) cells representing newly born neurons. (F) Nomarski optics image of the same panel shown in E.