Fig. 2.

Knockdown of Hand2 in cultured sympathetic neurons by siRNA. E12 sympathetic neurons were transfected with control GFP siRNA (A,B) or Hand2 siRNA (C,D) and analyzed after 2 days in culture for the expression of Hand2 mRNA by in situ hybridization. To control for the specificity of the signal obtained in the in situ hybridization also a sense RNA probe was used (E,F). Morphology of neurons and non-neuronal cells is shown in phase contrast (A,C,E), the in situ hybridization signal is shown in bright field optics (B,D,F). Hand2-positive neurons display a strong in situ hybridization signal (B,D), which is completely absent in the sense controls (F). Non-neuronal cells were also devoid of Hand2. In cultures treated with Hand2 siRNA only 44–50% of neurons were Hand2⁺, whereas >90% Hand2⁺ neurons were observed in control transfections. Data shown in (G) represent mean ± s.e.m. (n=3), **P<0,05.