Mineralization rate in zebrafish embryos treated with different LTG concentrations for 6 days. (A) 10 nM LTG treatment induces an increase of 71% of the mineralization rate respect the untreated embryos (CTR vs. 10 nM LTG, p < 0,05). The mineralization rate was evaluated as ratio of the number of mineralized vertebral bodies along body length (NV/L). (B) Alcian blue/ARS acid-free staining highlights an increased number of vertebral bodies (black arrows) in 10 nM LTG embryos vs untreated controls (CTR). No toxicity has been detected. . (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Calcein vital staining of scales from adult zebrafish treated with LTG ± PN evaluated for the presence of resorption lacunae (white arrow) along the scale border. The percentage indicates the fraction of the scales with that feature. PN-treated fish show resorption lacunae in all the scales while LTG+PN treated fish show an intact mineralized matrix in 90% of scales.

Analysis of TRAP activity in LTG-treated GIOP fish. (A) Percentage of TRAP-positive scales in adult zebrafish treated with different LTG concentrations with (PN vs 10pM LTG, p < 0.001; PN vs 100pM LTG, p < 0.001; PN vs 1 nM LTG) or without PN (CTR vs 10pM LTG, p < 0.001; CTR vs 100pM LTG, p < 0.001; CTR vs 1 nM LTG, p < 0.001; CTR vs 10 nM LTG, p < 0.001; CTR vs 100 nM LRG, p < 0,01). PN alone induced TRAP activity in 100% of scales (CTR vs 80 μM PN, p < 0.001) whereas 1 nM LTG suppressed PN-dependent TRAP activation (1 nM LTG + PN 80 μM vs PN 80 μM, p < 0.001, −90%). (B)Histochemical TRAP staining of scale treated with PN shows great resorption lacunae (purple staining) in all scales (100%) while only 10% of scales from 1 nM LTG+PN fish shows slight TRAP activity. (C) Biochemical TRAP analysis performed on scales confirms that 1 nM LTG treatment prevents the PN-dependent increase of TRAP activity (PN 80 μM vs CTR, p < 0.001, +99%; 1 nM LTG + PN 80 μM vs PN 80 μM, p < 0.001, −53%). (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Biochemical and histochemical ALP assay in scales of fish treated with PN ± LTG. (A) In the biochemical ALP assay, PN reduces ALP activity in all the scales whereas co-treatment with 1 nM LTG does not alter this effect (PN 80 μM vs CTR, p < 0.01, −38%; 1 nM LTG vs CTR, p < 0.01, −52%). (B) Histochemical ALP analysis confirms the PN-induced reduction of ALP activity (black arrows) is not modulated by 1 nM LTG. The percentage indicates the fraction of the scales with that feature.