Figure 4

HvNotch-knockdown transgenic Hydra. Vector pHyVec12 used for constitutive knockdown of HvNotch. (A) The Hairpin structure containing the sequence of HvNotch in sense and antisense orientation is under the control of the actin promoter. DsRed in the same operon is expressed independently after the addition of a splice leader. (B) Diagram presents the relative normalized expression of HvNotch, as determined by RT-qPCR with mRNA isolated from four HvNotch-knockdown strains (4#, 8#, 11# and 13#), three control strains (1#, 2#, 3#) injected with control-pHyVec12 and one empty polyp strain injected with HvNotch-hairpin-vector but without DsRed signals. HvNotch showed a significantly lower expression in Notch-knockdown strains in comparison with control groups (4#: p = 0.013; 8#: p = 0.0015; 11#: p = 0.0044; 13#: p = 0.0009). Primers were designed to amplify the extracellular domain of HvNotch sequences. (C) “Y-shaped” phenotype in strain 11# was observed in the initial stage (a, a’), the position of the joint moved into the foot region after one week (b, b’). a, b show light microscopy, a’, b’ show DsRed fluorescence.