a Alizarin red S/calcein live double staining in scales from fish of different ages (scale bar = 0.1 mm). Three-month-old fish showed a significant growth rate identified as thickness of the new deposited ring (green fluorescence). In 6-month-old fish scales, the thickness of new ring was found reduced, whereas 9-month-old fish did not show any significant deposition. b Quantification of the ring thickness in scales from 3-, 6- and 9-month-old fish (3 months vs 6 months, p < 0.001; 3 months vs 9 months, p < 0.001; 6 months vs 9 months, p < 0.001). The mineralization rate was inversely proportional to the age of the fish

a Anabolic activity on zebrafish scales measured by ALP biochemical assay normalized for scale area. No modulation of ALP activity was observed in 3-, 6- and 9-month-old fish. b Histochemical staining for ALP activity on aging scales (scale bar = 0.05 mm). A strong ALP activity has been detected in matrix-deposing cells (black arrow) in 3-month-old fish. The signal has become slightly downregulated in 6-month-old fish (black arrow) and disappeared in 9-month-old fish

a TRAP, measured by biochemical assay and normalized for scale area, has indicated a progressive increase of the activity in 3-, 6- and 9-month-old fish (3 months vs 9 months, p < 0.001; 6 months vs 9 months, p < 0.001). b Histochemical staining for TRAP activity on aging scales (scale bar = 0.1 mm). TRAP-positive signal has been detected along scale border of 9-month-old fish surrounding a bone resorption area. c Gene expression analysis on aging scales. Real-time PCR for bone-specific genes has shown an increase of tnfrsf1b (homolog of human rank) expression in scales from 6- and 9-month-old fish and an age-dependent downregulation of tnfsf11 and tnfrsf11b (homolog of human rankl and opg) (OPG: 3 months vs 6 months, p < 0.01; 3 months vs 9 months, p < 0.001; 6 months vs 9 months, p < 0.01. RANKL: 3 months vs 6 months, p < 0.01; 3 months vs 9 months, p < 0.001; 6 months vs 9 months, p < 0.01. RANK: 3 months vs 6 months, p < 0.001; 3 months vs 9 months, p < 0.001). d Calculated tnfsf11/tnfrsf11b (rankl/opg) ratio was found unchanged during aging

a Calculated ALP/TRAP ratio. A trend to bone resorption has been indicated by the lowering of ALP/TRAP ratio as the age increases (3 months vs 6 months, p < 0.05; 3 months vs 9 months, p < 0.001; 6 months vs 9 months, p < 0.001). b PTH level measured in fish blood showed that the hormone significantly increased with age (3 months vs 6 months, p < 0.001; 3 months vs 9 months, p < 0.001; 6 months vs 9 months, p < 0.001)

a Alizarin red/calcein live double staining of PN-treated scales from fish of different ages (scale bar = 0.1 mm). PN inhibited the formation of the new mineralized scale ring in fish of all ages and induced a significant bone resorption in 6- and 9-month-old fish scales (white arrows). b ALP and TRAP activity assay on PN-treated scales from fish of different ages. A significant reduction of ALP activity has been highlighted in 6- and 9-month-old fish treated with PN compared with untreated fish of the same age. The TRAP activity was significantly increased in PN-treated 9-month-old fish compared with earlier ages as well as with untreated fish of the same age (ALP: 6 months CTR vs 6 months PN, p < 0.001; 9 months CTR vs 9 months PN, p < 0.001; TRAP: 3 months CTR vs 9 months CTR, p < 0.001; 6 months CTR vs 9 months CTR, p < 0.05; 9 months CTR vs 9 months PN, p < 0.001). c Gene expression analysis for bone marker genes on PN-treated zebrafish scales. The expression of tnfrsf1b, tnfsf11 and tnfrsf11b (homologs of human rank, rankl and opg, respectively) was found increased in all ages after PN treatment (OPG: 3 months vs 9 months, p < 0.001; 6 months vs 9 months, p < 0.001. RANKL: 3 months vs 6 months, p < 0.001; 3 months vs 9 months, p < 0.001; 6 months vs 9 months, p < 0.001. RANK: 3 months vs 6 months, p < 0.001; 3 months vs 9 months, p < 0.001). d Calculated tnfsf11/tnfrsf11b (rankl/opg) ratio. The upregulation of tnfsf11 (human rankl) allowed the increase of the tnfsf11/tnfrsf11b (rankl/opg) ratio in 6- and 9 month-old PN-treated fish (3 months vs 6 months, p < 0.001; 3 months vs 9 months, p < 0.001). e ALP/TRAP ratio in PN-treated fish. Compared with the untreated fish, ALP/TRAP ratio decreased more rapidly during aging after PN treatment (6 months CTR vs 3 months CTR, − 15%, p < 0.05; 9 months CTR vs 3 months CTR, − 73%, p < 0.001; 9 months CTR vs 6 months CTR, − 68%, p < 0.001; 6 months PN vs 3 months PN, − 73%, p < 0.001; 9 months PN vs 3 months PN, − 90%, bp < 0.001; 9 months PN vs 6 months PN, − 18%, p < 0.01)

Comparison between bone growth (human male) and scale growth (zebrafish male) at different age. In zebrafish, the catabolic phase is accelerated by prednisolone (PN)