(A) In situ hybridizations to detect A-P tissue regionalization in control and ptk7(RNAi);wntP-2(RNAi) and ptk7(RNAi);ndl-3(RNAi) regenerating trunk fragments fixed 21 days after head and tail amputation, marking the anterior and head region (ndk, wnt2), prepharyngeal region (novel gene SMU15014980), trunk (novel gene SMU15007112, mmp1, foxA), posterior (wnt11-1, fzd4-1, Abd-Ba), and the anterior and posterior poles (wnt1, notum). All panels represent 100% of at least 6 animals stained. Arrow, ectopic trunk gene expression. Brackets, decrease in size of tail domain. (B) Quantitation of domain size changes from experiments described in (A), measured as length of domain normalized to body length. ptk7(RNAi);wntP-2(RNAi) and ptk7(RNAi);ndl-3(RNAi) regenerating trunk fragments had increased sizes of trunk domains marked by expression of mmp1, foxA and SMU15007112, and decreased sizes of tail domains marked by expression of wnt11-1 and fzd4-1 with little to no change to other domains. Asterisks, p<0.05 by 2-tailed t-test. (C) Both the pre-existing and ectopic pharynx in wntP-2(RNAi);ptk7(RNAi) animals regenerated (4/4 animals) after amputation with brief sodium azide treatment, using FISH to mark the gut (porcupine, green) and mouth and pharynx (laminin, red). Asterisk, pre-existing pharynx; arrows, ectopic pharynx, arrowhead, ectopic mouth. (C) ptk7(RNAi);wntP-2(RNAi) animals form ectopic FoxA+ cells by day 7 of regeneration. Bars, 100 (D), 200 (A), or 300 (C) microns.