(A, E, and K) Time course of crush-injury experiments in the larval abdominal ventral nerve cord (VNC), indicating the age of the larvae (after egg laying, AEL) when crush was applied (top arrows), followed by various recovery periods, and when they were dissected and fixed (bottom arrows). (C) Diagram showing that crush injury induced ectopic Dpn+ cells. (A, B, and D) Crush injury in the larval abdominal VNC at 74 hr AEL: (B) increased the levels of ia-2 mRNA at 5–7 hr post-injury, which recovered homeostatically by 24 hr, detected by qRT-PCR. N = 3 biological replicates. (D) Induced ectopic abdominal Dpn+ cells by 5–7 hr post-injury (74–80 hr AEL) in half of injured VNCs (penetrance 50%, N = 10), and increased (albeit not significantly) the total number of abdominal Dpn+ cells inthose samples. Mann–Whitney U-test, p=0.24. (F and L) Thoracic Ia-2YFP and Dpn signal were normal in thoracic neuroblasts, in samples shown in (G and H) and (M and N), respectively. (E–I) Injury at 96 hr AEL caused ectopic abdominal Dpn+ cells by 6 hr post-injury (arrowheads). Most Dpn+ cells were Ia-2YFP−. At this stage, some developmental neuroblasts could still remain (white arrowheads), but ectopic abdominal Dpn+ cells were dorsal (yellow arrowheads, H). Most injured VNCs had ectopic abdominal Dpn+ cells (88% penetrance N=9). (I) Unpaired Student's t-test p=0.0063. (J) Injury at 105 hr AEL and fixation at 129 hr AEL, when no developmental neuroblasts remain, induced a significant increase in abdominal Dpn+ cells (54.5% penetrance N = 11). Mann–Whitney U-test p=0.0375. (K–O) Injury at 117 hr AEL caused ectopic abdominal Dpn+ cells by 12 hr post-injury (129 hr AEL). . Ectopic abdominal Dpn+ cells were found in ectopic dorsal positions (yellow arrowheads, N). This stage is devoid of developmental neural stem cells. Over two thirds of injured VNCs had ectopic Dpn+ cells (67% penetrance N=21). (O) Student's t-test p=0.0302. (I,J,O) All abdominal Dpn+ cells were counted in all injured samples. (P) Temporal profile of number of ectopic Dpn+ cells surrounding the lesions, number in X-axis indicate time points of injury and fixation. (F, G, L, and M) Horizontal views, (H and N) transverse views. (D, I, J, O ) Graphs show box-plots . (P) Shows dot plots, with mean and error bars (±s.d.) indicated. *p<0.05, **p<0.01. For full genotypes and further statistical analysis details, see Supplementary file 1.