(A) Proliferation assay of single and double amputated trunk pieces at different times of regeneration indicate double cuts produce a decrease in the amplitude of both characteristic mitotic peaks and a delay in the first mitotic peak (mean number of mitotic cells, error bars are +/− 1 SEM of the mean, n = 12 at each time point). (B) Proliferation assay and(C) FACS analysis of the percentage of G2 cells in control (blue, n = 12) and HU (green, n = 10) treated regenerating pieces at different times of regeneration, confirms the efficiency of HU treatment to block pASCs progressing through S-phase of the cell cycle. Smed-GluR expression shows brain regeneration in (D,H,L) the anterior of control dsRed(RNAi), (E,I,M) Smed-APC-1(RNAi), (F,J,N) Smed-ptc(RNAi) and (G,K) anterior and posterior of Smed-βcatenin-1(RNAi) trunks pieces at 72 hR. Smed-GluR expression is observed in anterior blastemas of control dsRed(RNAi), Smed-APC-1(RNAi), Smed-ptc(RNAi) and anterior but not the posterior of Smed-βcatenin-1(RNAi) HU treated trunks at 72hR implicating pASCs that have progressed through S-phase at the anterior wound site as the source of cells for early brain regeneration. (L–N) Smed-GluR expression reveals brain regeneration in anterior blastemas of control dsRed(RNAi) but not Smed-APC-1(RNAi) and Smed-ptc(RNAi) double amputated trunks at 72hR after a second cut revealing a 16 hour window in which early Wnt/Hh independent brain regeneration occurs. (O,P,Q) Smed-GluR expression is shown in double amputated trunks at 40dR indicating that brain regeneration in control dsRed(RNAi), Smed-APC-1(RNAi) and Smed-ptc(RNAi) phenotypes can occur independently of the Wnt and Hh independent phase of the first 16 hours. Scale bars represent 100 µm in panels D, E, F, H,I ,J ,L, M and N and 250 µm in G and K and after 40 dR in O, P and Q.