(A) Hemiray removal scheme creating a proximal and distal facing injury on both sides of a central intact segment. (B) At 3 days post injury (dpi), Runx2+ preosteoblasts and committed osteoblasts expressing Osterix have only accumulated in the bone defect beyond the distal injury site of the centre segment, but not beyond the proximal injury site, as determined by immunofluorescence. Dashed line outlines centre segment. Scale bar, 10 µm. (C, D) Quantification of the number of Runx2+ cells (C) or Osterix+ cells (D) located in the bone defect beyond the proximal and distal injury sites of the centre segment at 3 dpi. N (experiments)=2, n (rays)=15. Error bars represent 95% CI. Wilcoxon matched-pairs test. (E) At 5 dpi, mineralised bone as detected by alizarin red staining has formed beyond the distal, but not the proximal injury site of the centre segment. Dashed line indicates centre segment. n=16/20 rays with distal bone formation. Scale bar, 100 µm. (F) Distribution of all positions along the proximodistal axis of centre segments, where proliferating EdU+ Zns5+ osteoblasts were observed at 2 and 3 dpi. Yellow dashed lines indicate segment border. Solid red lines, median; dashed lines, quartiles. N (experiments)=1, 2 dpi: n (rays)=13, n (cells)=306; 3 dpi: n (rays)=17, n (cells)=305; random set: n (groups)=12, n (points)=360. Kolmogorov-Smirnov test. (G) RNAscope in situ detection of bglap expression in the centre segment and the two adjacent segments distal to the bone defect at 1 dpi. Dashed lines indicate the proximal, central, and distal regions of the centre segment used for quantification. Bracket indicates dedifferentiation zone in the distal segments. Scale bar, 100 µm. (H, I) Single cell analysis of RNAscope intensity of bglap (H) or cyp26b1 (I) relative to the brightest signal in proximal, central, and distal regions of the centre segment at 1 dpi. N (experiments)=2, n (segments)=8 (bglap), 7 (cyp26b1). Error bars represent 95% CI. Holm-Sidak’s multiple comparison test. (J) Migration of bglap:GFP+ osteoblasts towards both injury sites of the centre segment. Yellow dashed lines, segment borders. Pink arrowheads indicate relocation of GFP+ osteoblasts. Asterisk indicates GFP+ osteoblasts that have entered the distal bone defect. Distal to the right. N (experiments)=4, n (segments)=95. Error bars represent 95% CI. Wilcoxon matched-pairs test. Scale bar, 100 µm. (K, L) Sequential hemiray injuries reveal no preference for osteoblast migration in distal vs proximal directions. Relocation of bglap:GFP+ osteoblasts in the centre segment (segment B) is plotted. Error bars represent 95% CI. Red arrowheads indicate time points at which the adjacent hemirays (A=proximal, C=distal) were removed. Negative relocation indicates increased distance between GFP+ cells and the joint. (K) Removal of the proximal adjacent segment A at 0 dpi, followed by removal of the distal adjacent segment C at 1 dpi. (L) Removal of the distal adjacent segment C at 0 dpi, followed by removal of the proximal adjacent segment A at 1 dpi. (M) Migration of osteoblasts into the bone defect at 3 dpi. Treatment with FK506 induces recruitment of GFP+ cells beyond the proximal injury site and increases the number of centre segments that show recruitment of GFP+ cells beyond the distal injury site. N (experiments)=1, n (segments)=56 (DMSO), 17 (FK506). Fisher’s test.