Fig. 3. The smedbmp4-1 pathway is needed for regeneration of a normal midline. (A) The diagram on the left shows the sites of amputations (black lines); data for trunks regenerating new heads and tails are shown. Animals were at 7 (unc-22 and smedbmp4-1 RNAi animals), 8 (smedolloid-1 and smedsmad4-1 RNAi animals) and 6 days (smedsmad4-1 prolonged RNAi) of regeneration. Yellow asterisks mark indented cephalic blastemas and red asterisks mark indented caudal blastemas. (B) Animals were labeled with the D.21.6 riboprobe that recognizes cells at the animal boundary (purple). Black arrows indicate cephalic and caudal blastema midline. The unc-22(RNAi) and smedolloid-1(RNAi) animals were at 11 days of regeneration, the smedsmad4-1(RNAi) animal was at 8 days of regeneration, and the smedbmp4-1(RNAi) animal was at 9 days of regeneration. (C) Prolonged exposure to smedsmad4-1 dsRNA completely blocked blastema formation. Animals were fed dsRNA on days 1, 5, 13 and 17 and amputated the following day. Animals were decapitated and had tails removed. Blastema formation failed in the smedsmad4-1(RNAi) animals at 6 days following amputation (middle). Outgrowths (arrows) of tissue from pre-existing tissue in a smedsmad4-1(RNAi) animal showed head-like structures at 16 days of regeneration (right). Scale bars: 0.1 mm. Anterior, left.