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Fig. 5
Differentiated scleroblasts from bone lining migrate to integrate in the blastema. (A-E) In vivo confocal images from a double transgenic Tg(eab2:EGFP-T-mCherry; osteocalcin:GFP) fish, injected with Cre mRNA. (A) Uncut fin; the arrows indicate yellow cells in the bone lining to be tracked upon amputation. (B) 12 hpa fin, the arrows indicate tracked yellow cells in the lining of the bone that have become rounder, indicative of their proliferative state. (C) 24 hpa fin; the yellow cells have migrated from the bone lining to the blastema. (D) 30 hpa fin. (E) 34 hpa fin. In D and E, the arrows mark yellow cells that progressively lose GFP expression driven by the osteocalcin promoter and expresses only mCherry. The sequential images (B-F) were taken from the same ray of the same transgenic fish, in total we tracked five animals. The images are a projection of several confocal optical slices. Scale bar: 50 μm in all images. Dashed lines indicate amputation plane. (F) A diagram showing the different possible outcomes of a cell from a double transgenic fish, Tg(eab2:EGFP-T-mCherry; osteocalcin:GFP) injected with Cre mRNA. An osteocalcin:GFP-positive cell in which the switchable marker (eab2:EGFP-T-mCherry) has not been activated is seen as a green cell. However, with the activation, the osteocalcin:GFP-positive cell also expresses mCherry, and is seen as yellow. Then, if the osteocalcin:GFP expression is downregulated the yellow cell can be seen as red. This red cell can turn yellow again when osteocalcin:GFP is re-expressed at 72 hpa (G) qPCR shows that at 12-48 hpa osteocalcin expression decreases compared with uncut fins and that at 72 and 96 hpa this expression increases sixfold and 23-fold, respectively (P<0.05, n=6 for each condition). (H-M) In vivo imaging of Tg(osteocalcin:GFP) from 19 to 20.5 hpa. The interval between each frame is 20 minutes. (H) A single ostecalcin-positive cell, artificially coloured green, is tracked in the subsequent images, I-M (in H the position of this cell was not clear). It is possible to see the cell extending protrusions, changing shape and migrating to integrate in the blastema. Another osteocalcin-positive cell at the bone lining is identified in I and is tracked in sequential images, J-M. The arrow in J indicates a proliferating osteocalcin-positive cell. Scale bar: 50 μm in all images. Arrowheads indicate amputation plane.