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Fig. 4
Action of later Fgf signalling on mesenchymal cells is required for cell proliferation. (A) Tracking of the behaviour of labelled cells from 2 to 5dpa. Normally (HS, i.e. normal Fgf signalling), the DsRed2-labelled mesenchymal cells increased in number and contributed to the entire region of a regenerate after 3dpa (n=9/9 successful transplants), whereas Fgf non-responsive mesenchymal cells rapidly retracted from the blastema in the newly regenerated region (+HS; n=8/8 successful transplants). White dashed lines, the site of amputation; yellow dashed lines, the distal end of the blastema at 2dpa and its corresponding sites at subsequent stages; brackets, the approximate area of the blastema (200µm from the distal tip). Scale bar: 200µm. (B) Detection of proliferating cells by EdU labelling at 4dpa. Labelling lasted for 6h before sampling. With intact Fgf signalling (HS), many DsRed2-positive (green) cells were located in the proliferation zone (brackets) and were also positive for EdU. With inhibition of Fgf signalling (+HS), most of DsRed2-positive cells were found outside the proliferation zone and were not showing EdU staining. The tissue sections were counter-stained with DAPI. Scale bar: 20µm. (C) Measurement of the proportion of EdU-positive cells among progenies of the transplanted cells. Approximately 30% of the cells were EdU-positive in the absence of heat shock, but this ratio decreased to 5% by the inhibition of Fgf signalling. n, the number of fin rays that successfully contained many DsRed2-positive mesenchymal cells and were used for quantification (five fish in total, respectively). Error bars represent meanħs.e.m. Student′s t-test was performed to assess statistical significance. **P<0.001. (D) Determination of the total number of EdU-positive cells in the regenerates in C. Overall cell proliferation was not different between the samples with (+HS) and without (HS) heat shock. Error bars represent meanħs.e.m. Student′s t-test was performed to evaluate statistical significance. n.s., not significant.