Endochondral ossification, the process by which most of the bone is formed, is regulated by many specific groups of molecules and extracellular matrix components. Hind limb of Bufo gargarizans is a model to study endochondral ossification during metamorphosis. Chinese toad (Bufo gargarizans) were exposed to different fluoride concentrations (0, 1, 5, 10 and 20 mg L⁻¹) from G3 to G42. The development of hind limb of B. gargarizans was observed using the double staining methodology. The transcriptome of hind limb of B. gargarizans was conducted using RNA-seq approach, and differentially expressed gene was also validated. In addition, the location of Sox9 and Ihh in the growth cartilage was determined using in situ hybridization. Our results showed that 5 mg L⁻¹ stimulated bone mineralization, while 10 and 20 mg L⁻¹ exposure could inhibit the tibio-fibula, tarsus and metacarpals ossification. Besides, 10 mg F/L treatment could down-regulate Ihh, Sox9, D2, D3, TRα, TRβ, Wnt10, FGF3 and BMP6 expression, while up-regulate ObRb and HHAT mRNA expression in the hind limb of B. gargarizans. Transcript level changes of Ihh, Sox9, D2, D3, TRα, TRβ, Wnt10, FGF3 and BMP6 were consistent with the results of RT-qPCR. In situ hybridization revealed that Ihh was expressed in prehypertrophic chondrocytes, while Sox9 was abundantly expressed in proliferous, prehypertrophic and hypertrophic chondrocytes. However, 10 mg F-/L did not cause any affect in the location of the Ihh and Sox9 mRNA. Therefore, high concentration of fluoride could affect the ossification-related genes mRNA expression and then inhibit the endochondral ossification. The present study thus will greatly contribute to our understanding of the effect of environmental contaminant on ossification in amphibian.

软骨内骨化是大部分骨骼形成的过程，受分子和细胞外基质成分的许多特定基团调节。中华大蟾蜍的后肢是研究变态过程中软骨内骨化的模型。从G3到G42，大蟾蜍（Bufo gargarizans）暴露于不同的氟化物浓度（0、1、5、10和20 mg L ^-1）。使用双重染色方法观察到了B. gargarizans的后肢发育。使用RNA-seq方法对B. gargarizans的后肢进行转录，并验证了差异表达的基因。此外，使用以下方法确定了Sox9和Ihh在生长软骨中的位置：原位杂交。我们的结果表明，5 mg L ^-1刺激骨矿化，而10和20 mg L ^-1暴露可抑制胫腓骨，骨和掌骨骨化。此外，10 mg F / L处理可下调I.hh，Sox9，D2，D3，TRα，TRβ，Wnt10，FGF3和BMP6的表达，而上调B. gargarizans后肢的ObRb和HHAT mRNA表达。Ihh，Sox9，D2，D3，TRα，TRβ，Wnt10，FGF3和BMP6的转录水平变化与RT-qPCR结果一致。原位杂交显示，Ihh在肥大前的软骨细胞中表达，而Sox9在增生，肥大和肥厚的软骨细胞中大量表达。但是，10 mg F- / L不会对Ihh和Sox9 mRNA的位置产生任何影响。因此，高浓度的氟化物可能影响骨化相关基因的mRNA表达，进而抑制软骨内骨化。因此，本研究将大大有助于我们理解环境污染物对两栖动物骨化的影响。