Fig. 1. AQP4 is required for ANSC self-renewal and proliferation in vitro. (A) Morphological examination showed the reduced number and size of Aqp4^–/– primary, secondary and tertiary neurospheres compared with the Aqp4^+/+ control. Primary neurospheres were initiated from dissociated adult rostral periventricular tissue. Cells obtained by dissociating primary neurospheres proliferated and yielded secondary neurospheres. Single cells obtained after dissociation of secondary neurospheres proliferated and yielded tertiary neurospheres. Scale bar: 200 µm. (B,C) Neurosphere assay. The number and size of neurospheres were determined after 7 days for primary, secondary and tertiary neurospheres (^*P<0.05 vs Aqp4^+/+, n=3). (D,E) BrdU incorporation assay. The percentage of BrdU⁺ cells in Aqp4^–/– ANSCs was significantly lower than that in Aqp4^+/+ cells (^*P<0.05, n=4).