Electroporation of dominant negative RARs abolishes CdxA expression and pancreas formation.
Electroporation of pCIG (A,B,G,I,K) or pCIG-DNRAR (C–F,H,J,L). Whole mount in situ hybridization on stage HH 19 embryos shows CdxA expression (blue) in the closed duodenum and the open midgut. CdxA expression is repressed either partially (n = 7/11, C) or completely (n = 3/11, F) by DN-RAR. Cells expressing the expression construct are labeled by subsequent immunocytochemistry for GFP expressed from the bicistronic construct (Green in D, F and H, masked by blue CdxA staining in B, DAB-brown in G), demonstrating that repression extends to the neighbors of targeted cells. Whole mount immunocytochemistry on stage HH 20 embryos shows that dominant negative RAR (traced with GFP, green) represses pancreas progenitor emergence (traced with Nkx6.1, red) in a non-cell autonomous manner (J) as compared to control embryos electroporated with empty vector (I). Glucagon+ cells could still differentiate (blue). (K and L) Selected optical sections of embryos displayed in (I) and (J), respectively. Scale bar 200 µm.