Molecular Mechanism Of Tail Uptake In Bufo Gargarizans And Rana Chensinensis

Amphibian metamorphosis is a postembryonic development process precisely regulated by thyroid hormones.During this postembryonic development,a number of physiological and biochemical processes will take place,causing changes in the tissues and organs of the tadpole,such as tail absorption.The tail absorption of an amphibian tadpole is an important event in the metamorphosis of amphibians.In the process of tail absorption,the tail of the tadpole is gradually absorbed,and the movement mode of the tadpole will change from tail swimming to limb movement.From an evolutionary perspective,this has important implications for the adaptation of amphibians from aquatic life to terrestrial life.In the process of tadpole tail absorption,not only morphological changes were carried out in the tail,but also complicated physiological and biochemical processes as well as multiple cellular processes were conducted.In these processes,apoptosis and autophagy have been shown to play an important role in the tail absorption of various amphibians,and are one of the molecular mechanisms of tail absorption in amphibians.However,the molecular mechanism of tail absorption is not clear in the Bufo bufo gargarigans and Rana chensinensis,and whether apoptosis and autophagy are involved in tail absorption or their role in the tail absorption is still unknown.Therefore,we investigated the tail absorption process of Bufo bufo gargarigans and Rana chensinensis in order to understand the molecular mechanism of tail absorption of the Bufo bufo gargarigans and Rana chensinensis.In this study,morphological,histological,real-time quantitative PCR(RT-qPCR),transcriptome sequencing and other methods were used to explore the molecular mechanism of the tail absorption process of Bufo gargarigans and Rana chensinensis.This study is divided into two parts.In the first part,we studied the absorption process and its molecular mechanism in the tail of the Bufo gargarigans.We first observed and photographed the morphological changes of the tail of the tadpole of the Bufo gargarigans at the Gs38-46 stage,measured and recorded the tail length of the tadpole at the Gs38-46 stage,and then carried out histological staining of the tail of the Gs38,42,44 stage through HE and Masson staining to observe the histological changes of different tissues during the absorption process of the tadpole tail.In addition,we also screened and analyzed the differentially expressed genes in the tail and hind limbs of Rana chensinensis through transcriptomics sequencing and RT-qPCR technology to obtain the candidate genes,and measured the expression level of the candidate genes in the tail of the tadpole of Gs38,42,44 by RT-qPCR.In the second part,we explored the tail absorption process and its molecular mechanism.By observing and photographing the tail of Rana chensinensis,we recorded the morphological changes of the tail absorption process of Rana chensinensis,and measured the tail length of tadpole at Gs38-46 stage.Subsequently,histological changes in the tail of Rana chensinensis tadpole of Gs38,42,44 were observed by HE and Masson histological staining.Finally,using transcriptomic-sequencing technology,we screened and analyzed the differentially expressed genes in the tail and hind limbs of Rana chensinensis,and obtained candidate genes.In addition,we compared the tail absorption processes of the Bufo gargarigans and Rana chensinensis to assess the similarities and differences of the tail absorption processes between the two species.The conclusions are as follows:1.Our morphological measurement results showed that the tail absorption rate of the tadpoles of Bufo gargarigans and Rana chensinensis was slow before the peak of metamorphosis,and suddenly accelerated after reaching the peak of metamorphosis at Gs42 stage,the tail of the tadpoles at Gs46 stage was completely absorbed.2.The histological results of HE and Masson staining showed that fast muscle and slow muscle of the tail of Bufo gargarigans and Rana chensinensis were absorbed by apoptosis in the process of tail absorption,fast muscle began to disappear at Gs40 stage,while slow muscle began to apoptosis after Gs40 stage,and melanin macrophages played an important role in the process of muscle apoptosis.In addition,histological staining of the notochord showed that fibroblasts first invaded the notochord,causing notochord shrinkage,and then entered the notochord sheath for degradation.the area of muscle cells decreased during the process of absorption of the tail of Bufo gargarigans,and obvious apoptotic degradation.3.Transcriptome sequencing results showed that there were many differentially expressed genes in the tail and hind limbs of the tadpoles of Bufo gargarigans and Rana chensinensis.We analyzed and screened the differentially expressed genes.We found that apoptosis related genes CASP6 and APAF1 and autophagy related genes ATG5 and ATG7 were significantly up-regulated.The results showed that apoptosis and autophagy were involved in the tail absorption process.4.The histological section of the thyroid gland of tadpoles at Gs38,42 and 44 stages showed that the height of follicular epithelial cells was the highest and the thyroid activity was the highest at Gs42 stage,suggesting that the thyroid secretion level of thyroid hormone reached the peak.Gs38 and Gs44 have low thyroid secretion.5.Real-time quantitative PCR(RT-qPCR),according to the results in the abnormal process,compared with Gs38 stage,CASP6 Gs42 stage of apoptosis related gene CASP6 and APAF1,autophagy related genes ATG5 and ATG7,cell component degradation related genes MMP9 and TH receptors TR? and iodine enzyme D2 were significantly higher,and oxidative stress related genes GPX,SOD,HSP90,iodine enzyme D3 and TH receptor TRa lowered significantly.The results showed that apoptosis,autophagy and degradation of cellular components were involved in tail absorption.However,oxidative stress-related genes in the tail of tadpoles were significantly down-regulated and antioxidant capacity was decreased.The decreased antioxidant capacity of tadpole tail tissue can lead to the accumulation of reactive oxygen species,causing cell death.Therefore,we speculated that oxidative stress might be involved in the tail absorption process of tadpoles.In conclusion,the tail absorption process of Bufo gargarigans and Rana chensinensis is a complex process,which is co-regulated by apoptosis,autophagy,cell component degradation and oxidative stress,and TH receptor and deiodase can also affect the tail absorption process of tadpoles.This study provides experimental and theoretical basis for further study on the tail absorption process of tadpole of Bufo gargarigans and Rana chensinensis.