| Fluoride is widely distributed in nature in many forms except its free form and exists only in combination with other elements as fluoride compounds.In natural waters fluorine is commonly a minor component,with contents usually lower than 1 mg/l.However,the concentration of Fluoride has increased dramatically due to anthropogenic activities,such as phosphate fertilizers,synthetic pigments,semiconductor manufacturing,glass and brickmaking industries,and coal power plants.Fluoride is considered one of the most essential elements for human and animal health.However,high concentrations of fluoride can cause damage to multiple organs and tissues especially to the skeleton,progressing to fluorosis.Bone plays important roles in forming the skeleton,organ protection and mineral storage.Bone develops in two different ways:intramembranous ossification and endochondral ossification.Endochondral ossification,the process by which most of the skeleton is formed,consists of three steps,including proliferation of chondrocytes,hypertrophy of chondrocytes and maturation of cartilage matrices.This complex biological event is regulated by many specific groups of molecules and extracellular matrix components.Systemic factors,transcription factors and hormones are involved in this complex regelation of endochondral ossification.Systemic factors could regulate the behaviour of chondrocytes in growth cartilage,include growth hormone(GH)and thyroid hormone(THs),and transcription factors include SRY-box containing gene9(Sox9)and Myocyte enhancer factor-2(MEF2C).In addition,the local secreted factors include Indian hedgehog(Ihh),parathyroid hormone-related peptide,fibroblast growth factors(FGFs).Thyroid hormones(THs)has been known for many years to be an important regulator of skeletal growth.THs action in bone is mediated mainly via thyroid hormone receptor a(TRa),which is expressed in the skeleton at>10-fold higher levels than thyroid hormone,receptor β(TRβ).As a member of locally secreted factors,Indian hedgehog(Ihh)protein plays a key role in endochondral ossification in mammals.Ihh predominantly expressed in prehypertrophic chondrocytes in the growth plate at early stage of endochondral ossification.Sox9 is initially expressed in mesenchymal condensations,and early expression is necessary for further differentiation of chondrocytes.The multiple function of Sox9 is from mesenchymal condensation to chondrocyte differentiationAlthough numerous studies focused on the molecular mechanisms of skeletal tissue development in mammals,relatively little is known about the molecular processes of endochondral ossification in amphibian.Therefore,study that could gain new insight into the molecular mechanisms of endochondral ossification and that uncover the effect of fluoride on endochondral ossification in amphibian is quite meaningful.Endochondral ossification of limb in amphibian occurs in postembryonic development.Thus,the effect of fluoride on endochondral ossification can be researched though the treatment of different fluoride concentrations.In the present study,the larva of B.gargarizans was used to study the molecular mechanisms of endochondral ossification and the effect of fluoride on endochondral ossification in amphibian.In the first part of the present study,the development of hind limb of B.gargarizans were observed using the double staining methodology from Gs40 to Gs46.Then transcriptome of hind limb of B.gargarizans were conducted by using RNA-seq approach and developmental expression profiles of genes related to endochondral ossification were investigated over different developmental stages(Gs40-Gs46).In situ hybridization was also conducted to study the location of Sox9 and Ihh in the growth cartilage.In the second part,Chinese toad(Bufo gargarizans)were exposed to different fluoride concentrations(0,1,5,10 and 20 mg L"1)from Gs3 to Gs42.The development of hind limb of B.gargarizans were also observed using the double staining methodology.The transcriptome of hind limb of B.gargarizans were conducted by using RNA-seq approach,and differentially expressed gene was validated by using RT-qPCR.In addition,the location of Sox9 and Ihh in the growth cartilage was also determined using in situ hybridization.Main results are as follows:1.The double staining results indicated that the hind limb was ossified gradually and was ossified from proximal to distal in B.gargarizans during metamorphosis.The femur,the tibio-fibula and tarsus began to ossify at Gs40,while they were ossified completely at Gs42.In addition,the whole hind limb bone was fully ossified at Gs46.This indicate that endochondral ossification of hind limb of B.gargarizans occours during metamorphosis,and that hind limb is an excellent model to study endochondral ossification.2.31 genes related to endochondral ossification were found in the cDNA library of hind limb by using RNA-Seq technology,and the expression profiles of these genes were analyzed during the metamorphosis.In addition,9 of these genes were validated by RT-qPCR.It indicates that the results of the present study are reliable.3.Based on the results of in situ hybridization,the Sox9 was expressed in the columnar,prehypertrophic,and upper hypertrophic zones,while the expression of Ihh was observed in prehypertrophic chondrocytes zone in hind limb of B.gargarizans.Ihh play important roles in the early stage of endochondral ossification,while the multiple function of Sox9 is from mesenchymal condensation to chondrocyte differentiation4.10 mg F-/L exposure could inhibit the endochondral ossification in hind limb of B.gargarizans by using double-staining technique.For.example,the tarsus including fibulare and tibiale began to ossify in control group,whereas the tarsus was no ossified with obvious cartilage in 10 mg F-/L group at Gs40.5.10 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 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.Thus,fluoride could affect the ossification-related genes mRNA expression.6.In situ hybridization revealed that 10 mg F-/L did not cause any affect in the location of the Ihh and Sox9 mRNA in hind limb of B.gargarizans.This indicate that fluoride did not affect the location of ossification-related genes mRNA expression.In conclusion,endochondral ossification requires coordinated interplay of many specific groups of molecules.Hormones,systemic factors and transcription factors are involved in the three stages of endochondral ossification.In addition,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 endochondral ossification and will provide.a valuable genomic resource and background for the future study of endochondral ossification in amphibian.Moreover,the present study will greatly be conducive to our understanding of the effect of environmental contaminant on ossification in amphibian. |
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