- Title
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Zebrafish fin regeneration involves generic and regeneration-specific osteoblast injury responses
- Authors
- Sehring, I.M., Mohammadi, H.F., Haffner-Luntzer, M., Ignatius, A., Huber-Lang, M., Weidinger, G.
- Source
- Full text @ Elife
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(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. |
Osteoblast dedifferentiation and migration represent generic injury responses that are differentially regulated and can occur independently of each other and of regenerative bone growth. Upper panel: After fin amputation, osteoblasts downregulate the expression of differentiation markers. The extent depends on their distance to the amputation site, with osteoblasts close to the amputation site displaying more pronounced dedifferentiation. In addition, osteoblasts elongate and migrate towards the amputation plane and beyond to found osteogenic cells in the blastema (pink). Thus, osteoblast dedifferentiation, migration, and bone regeneration are all distally oriented. Osteoblast dedifferentiation is negatively regulated by NF-κB and retinoic acid signalling, while actomyosin dynamics and the complement system are required for directed osteoblast migration. Lower panel: In the hemiray removal model, a proximal and a distal injury are created on both sides of a remaining centre segment. The extent of osteoblast dedifferentiation is even along the centre segment, and osteoblasts migrate towards both injury sites. Yet, only at the distally-facing site, osteoblasts migrate into the bone defect, and blastema formation and bone regeneration only occur here. |