| Amphibians are the first animals to have limbs in biological evolution.The emergence of limbs helps amphibians move on land and adapt to land life.The movement of different amphibian species is closely related to the length of forelimbs and hindlimbs:individuals that normally move by jumping usually have relatively longer hindlimbs and shorter forelimbs,while species that walk-jump in the ground generally have relatively longer forelimbs.Jumping is a unique form of locomotion for anuran,which helps individuals escape from predators.Therefore,the survival condition of anuran is dependent on the jumping performance.However,there are few studies on the comparison of jumping performance between anuran species.Jumping ability is found to depend on relative hindlimb length and relative hind limb muscle weight.In addition,osteoarticular changes in the hindlimb/pelvis morphometrics,iliac-sacral and sacro-urostylic joints are also proposed to be a key role in jumping.The presence of bone constructed a framework for the architecture of the limbs and provided attachment sites for muscles.The formation of appendicular bone is mainly through the process of endochondral ossification,which is strictly regulated by a network of gene interactions managed both temporally and spatially.These genes in mammalian mainly include the following three categories:system factors represented by thyroid hormones(THs),transcription factors represented by SRY-box containing genes(Sox),and local secretory factors represented by fibroblast growth factors(FGFs)and bone morphogenetic proteins(BMPs).The first section of this paper compared the jumping ability of the young frog(Gs 46)in Rana chensinensis and Bufo gargarigans.Firstly,the average maximum jump distance and morphological parameters of the two species were measured.Secondly,the ossified degree of the two species was compared by the double staining method of bone,and the bone sections of the hindlimbs were observed by hematoxylin-eosin(H?E)and Masson tricolor staining methods.The second part took Rana chensinensis tadpoles as the samples to explore changes of structure related to the jumping ability and the molecular mechanism during the development process.Morphological changes of Rana chensinensis were determined by morphological methods during the development from Gs 40 to Gs 46.The degree of bone development was observed in tadpoles at Gs 40,Gs 42 and Gs 46 three determined stages.The changes of hindlimbs sections and thyroid gland were also observed at Gs 40,Gs 42 and Gs 46 with H?E and Masson tricolor staining methods.In addition,transcriptome sequencing technology was used to analyze the expression levels of genes related to endochondral ossification in the hindlimbs of Rana chensinensis at Gs 40,Gs 42 and Gs 46.The results and conclusions are as follows:The first section:The average maximum jumping distance of Rana chensinensis was greater than that of Bufo gargarizans,which indicated that Rana chensinensis had better jumping ability than that of Bufo gargarizans.Morphological analysis showed that the snout-vent-length,hindlimb length and body weight of Rana chensinensis was higher than Bufo gargarizans.The results of double bone staining showed that the degree of ossification of Rana chensinensis was higher than that of Bufo gargarizans,in which the urostyle of Rana chensinensis had fused with the posterior sacral vertebrae and showed a complete rod-like structure,while the Bufo gargarizans had not fused with the postsacral vertebra.The results of bone section staining showed that the fused tendency of the tibial and fibular cortical bone in Rana chensinensis was much higher than that in Bufo gargarizans.These structural differences may account for the difference in jumping ability between Rana chensinensis and Bufo gargarizans,and the higher level of skeletal structure development will be conducive to the enhancement of individual jumping ability.The second section:The bone length and ossification of Rana chensinensis are increasing continuously during the development process.The urostyle and the cortical bone of tibia-fibula were fully developed and fused at Gs 46.The histological changes were observed in thyroid tissue area and follicular epithelial cell height during development,which were an adaptation for different THs demand levels in bone development.In addition,the transcriptional analysis of ossification related differentially expressed genes was similar to that in mammals,suggesting that the regulation of endochondral ossification related genes in amphibians may be consistent with that in mammals.The difference of expression level of ossification related genes in different developmental stages indicated that endochondral ossification of hindlimbs increased gradually during development.These changes may provide greater mechanical strength for the bones of Rana chensinensis,enhancing the locomotion ability and thus providing further support for the movement of amphibians on land.In summary,this study compared the average maximum jumping distance between Rana chensinensis and Bufo gargarizans,and studied the external morphological characteristics,internal bone structure and gene expression regulation level related to jumping ability by using morphological and histological methods.The results showed that the jumping ability of Rana chensinensis was better than that of Bufo gargarizans.The excellent jumping ability of Rana chensinensis was inseparable from its external morphological characteristics and the structural integrity of its urostyle and tibiofibula,which was gradually improved in the development process.Furthermore,the development of hindlimb bones was continuously regulated by the expression of genes related to endochondral ossification at the molecular level.These results will help us to understand the physiological mechanism of amphibian jumping and provide a theoretical basis for understanding the life and survival ability of amphibians. |
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