Background Endochondral ossification, the process by which most of the skeleton is formed, is accurately regulated by many specific groups of molecules and extracellular matrix components. The molecular mechanisms of endochondral ossification have been extensively investigated in mammals. However, there are few studies about it in amphibians. Results Ossification of femur and tibiofibula was observed at Gs 40, and tarsals and metatarsals were ossified at Gs 42. Most of the skeletons in hindlimbs were completely ossified except for articular cartilages at Gs 46. Thirty-two genes related to endochondral ossification were found in the differentially expressed genes (DEGs) library of hindlimbs, and nine of these genes were validated by qRT-PCR during metamorphosis. Sox9 was expressed in the columnar, prehypertrophic, and upper hypertrophic zones, and the expression of Ihh was observed in prehypertrophic chondrocyte zone in hindlimbs of B. gargarizans. Conclusions The ossification of hindlimbs increased gradually, and the ossification sequence was from proximal to distal in B. gargarizans during metamorphosis. Thirty-two genes found in the DEGs library were related to the regulation of endochondral ossification of hindlimbs in amphibians. The present study will provide a valuable genomic resource for the future study of endochondral ossification in amphibian. Developmental Dynamics 247:1121-1134, 2018. © 2018 Wiley Periodicals, Inc.

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In the present study, responses of the Chinese brown frog (Rana chensinensis) to exposure to di-2-ethylhexyl phthalate (DEHP), a common plasticizer, during the larval period were characterized.The effects of DEHP on metamorphosis rate, thyroid hormone, thyroid histology and the expression of genes involved in the steroid hormone synthesis in gonad were investigated.Metamorphosis rate and 50 percent of the tadpoles to reach Gosner stage 42 (T 0.5 ) were significantly slower in all DEHP groups. The thyroid glands of the tadpoles exposed to DEHP clearly exhibited colloid depletion. In addition, decreased concentrations of T4 and T3 were observed in the tadpoles exposed to DEHP. Moreover, the highest DEHP exposure (10 mmol/L DEHP) showed increased ratio of females significantly. Also, up-regulation significantly of transcripts of cytochrome P450 aromatase (CYP19) gene was detected in male tadpoles exposed to DEHP. The present results indicate that this increase in estrogens could lead to female-biased sex ratio in DEHP exposure group. Taken together, the present study indicates that DEHP disrupt thyroid hormone and sex steroid signaling in R. chensinensis tadpoles. Our present observations support evidence of a crosstalk between TH and sex steroids in gonad differentiation.di-2-ethylhexyl phthalate, gonadal differentiation, metamorphosis, thyroid hormone, sex steroidrelated genes | I N TR ODU C TI ONOver the past decades, amphibian populations are globally decreasing at an alarming rate. In most cases, amphibian decline is attributed to the anthropogenic alterations of the environment including environmental pollutants, fertilizers and plasticizer. 1 DEHP (di-2-ethylhexyl phthalate), as a common plasticizer, is widely used in consumer products such as footwear, shower curtains and toys, medical devices, and commercial/ industrial uses such as resilient flooring, wall covering, roofing, aluminum foil coating/laminating, paper coating, extrudable molds and profiles, electrical component parts, and wire and cable coating. 2,3 Due to its widespread use, it is ubiquitously detected in the surface water and groundwater, drinking water, air, soil and plants and animals are widely detected. 4 The DEHP concentration of the surface water in Jiangsu area of the Yangtze river delta and in four sampling points of the Tianjin section of the Haihe River reached 28.403 lg/L and 101.1 lg/L, respectively. 5,6 These have exceeded the limit of DEHP concentration of China's surface water standard (8 lg/L). Moreover, the recent research showed that DEHP is found in poultry food, vegetables, juice, even the breast milk of sucking mother. 7 Therefore, the potential health effects of DEHP are receiving growing public concern.Many studies have demonstrated that DEHP is an endocrine disrupter, which interferes with thyroid hormone signaling. For example, an in vitro study showed that DEHP enhances iodine uptake by modulating the sodium/iodide symporter in rat thyroid cell line. 8 In addition, it has recently been reported that altered...

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