Fig. 4. Silencing of Hand2 prevents the in vitro development of enteric neurons. (A) Crest-derived cells immunoselected from E13 mouse gut, cultured and transfected with siRNA^Hand2/GFP or siRNA^scrambled/GFP. siRNA^Hand2/GFP interfered with the development of Hu+ neurons (pink). Notice the GFP-fluorescent axons (green) of the Hu+ neurons in cultures transfected with siRNA^scrambled/GFP (A; left), which are absent in those transfected with siRNA^Hand2/GFP (A; right). (B) Quantitative results. Overall density both of Hu+ neurons and transfected cells developing as neurons (doubly labeled with GFP and Hu) in cultures transfected with siRNA^Hand2/GFP are lower than those seen in cultures transfected with siRNA^scrambled/GFP. siRNA^Hand2/GFP does not differ from siRNA^scrambled/GFP in transfection efficiency measured on non-neuronal cells. Scale bar: 50 µm.