| In this study,the life history of Chinese concave-eared frogs(Odorrana tormota),which is unique to China,was taken as the main line,and the characteristics of the development and the mechanism of the molecular regulation of hindlimb thigh muscle in different life stages were discussed by synthetically using the principles and methods of morphology,developmental biology,histochemistry,transcription histology and epigenetics.The purpose of this study is to reveal the pattern of the development of hindlimb thigh muscle of this species,the difference between genders and its adaptability to the critical life-history stages(the metamorphosis stage,hibernation stage,etc).The main results are as follows:1.The individual growth and development of O.tormota(1)Body length,body weight,hindlimb length and hindlimb weight showed an increasing trend from the G26 stage(hindlimb bud)to adult for both genders,but the body weight decreased during metamorphosis.All indexes of the body gradually increased after the metamorphosis to the pre-hibernation.The weight and hindlimb weight decreased significantly during hibernation.After hibernation,the growth rate of each index accelerated,and the growth rate decreased gradually after 2 years of age.(2)From the beginning of the metamorphosis period,there were differences in body length,body weight,hindlimb length and hindlimb weight between genders,and all the indexes of the females were slightly higher than that of the males.At the age of 14 months,there were significant differences in physical indexes between genders,and indexes of the females were significantly larger than that of the males.2.The development characteristics of hindlimb thigh muscles in different life-history stages(1)General pattern of the development of hindlimb thigh muscles: stages from G26 to G35 were the proliferation phases for myogenous cells.stages from G36 to G41 were the phases for muscle tube fusion,where 11 muscle groups had formed at the G41 stage.Stages from G42 to 10 months of age were for the differentiation of muscle tubes and the formation of muscle fibers.After 14 months of age,muscles existed in the form of muscle fibers.It was found that the growth mode of hindlimb thigh muscle group were different in different species of frog family.(2)Gender differences in the diameter and quantity of muscle fibers in hindlimb thigh muscle groups: six muscle groups in hindlimb thigh muscles of females began to appear primary muscle fibers in the G42 stage while only 4muscle groups of males had primary muscle fibers.The total number of muscle fibers in the male frogs from G43 stage to 3-month-old was more than that of the females,and the total number of muscle fibers of the female frog after5-month-old was more than that of the male frogs.There were different differences in the diameters of the muscle fibers in different muscle groups before the age of 14 months,indicating the existence of the sexual type of hindlimb thigh muscles during the metamorphosis and froglet stages.(3)Changes in the number of muscle fibers in different muscle groups during metamorphosis and hibernation stages: the number of muscle fibers in 6and 5 muscle groups was decreased respectively in females and males for the process of metamorphosis.During hibernation,muscle fibers of three muscle group decreased in the hindlimb thigh of the females,while muscle fibers in 6muscle group decreased in the males.It is suggested that both genders can obtain energy by decomposing the muscle fibers of specific muscle groups to pull through the special life-history stages for metamorphosis and hibernation periods.3.Analysis of m RNA expression profiles of hindlimb thigh muscles at different developmental stages Hindlimb thigh muscles at different developmental stages(muscle cell existence,myotubes existence,pre-and post-metamorphism,pre-hibernation,hibernation,period of obvious difference in body size between genders,sexual maturity stage)were used to analyze m RNA expression profiles.(1)Differentially expressed genes related to associated with the development of hindlimb thigh muscles in different life-history stages by GO enrichment.There were 11482 differentially expressed genes between the G36 and G40 groups.141 differentially expressed genes,such as Myo D,MYF6,and OBSCN,related to muscle growth and movement were screened by GO enrichment.The up-regulated genes in the G40 stage may relate to the fusion and differentiation of myotubes in hindlimb thigh muscles.There were 8352 differentially expressed genes between G40 and G42 stages.149 differentially expressed genes related to muscle growth and movement,such as MYF6,MEF2 D,and FLOT2,etc,were screened by GO enrichment.The up-regulated genes in G42 stage may be related to myotubes differentiation and primary muscle fiber formation.Moreover,genes related to blood oxygen transport,glycogen and energy metabolism were up-regulated in G42.These genes may be related to the maintenance of energy supply(no food obtained)during metamorphosis and the enhancement of energy metabolism during the transition from water environment to terrestrial environment.There were 1254 differentially expressed genes between G42 and G45 stages,among which 55 differentially expressed genes related to muscle development and movement,such as CSRP3,and NPC1.The up-regulated genes in G45 may be associated with the proliferation or differentiation of muscle fibers in hindlimb thigh muscles.There were 1911 differentially expressed genes between G45 and3-month-old groups,among which 49 differentially expressed genes related to muscle growth and movement such as SYNE1 and TTN.The up-regulated genes at 3 months of age may be related to muscle fibers formation or hypertrophy in hindlimb thigh muscles.Moreover,9 genes related to glycogen metabolism items were up-regulated at G45,suggesting the body supplied glycogen by degradation of some muscle fibers of hindlimb thigh muscles to meet the needs of energy metabolism in this process.There were 6865 differentially expressed genes between 3-month-old and5-month-old groups,among which 144 differentially expressed genes related to muscle development and movement,such as FLOT2 and PDGFRA.The down-regulation of differential expression genes related to oxygen transport and binding,glycogen synthesis and glucose metabolism and mitochondria and energy metabolism in deep hibernation period suggested that their body was adapted to low temperature for up to 4 months by reducing their own energy metabolism.Furthermore,differentially expressed genes related to protein degradation in lysosome and ubiquitin metabolic pathways up-regulated at the age of 5 months(deep hibernation period)may promote protein degradation.Compared with the early hibernation,the muscle fibers of six hindlimb thigh muscle groups were significantly reduced during deep hibernation.These muscle fibers may be degraded to produce energy through lysosome and ubiquitin pathway,so as to meet the energy needs of the body to survive hibernation successfully.There were 5050 differentially expressed genes between 14-month-old and2-month-old groups,among which 192 differentially expressed genes related to muscle fiber maturation,regeneration and movement ability of hindlimb thigh muscles,such as CAPZA1,MEF2 D and FBXO40.There were 8032 differentially expressed genes between genders at 14 months of age,among which 107 differentially expressed genes related to muscle development and movement,such as HES4,SIX4 and MYOD-1A.72 genes related to protein synthesis and 50 genes related to protein degradation were all up-regulated in females,expressing promotion effect.In addition,genes related to glycogen synthesis and glucose metabolism were all up-regulated in females.These data suggested that large female individuals have strong material turnover,energy storage and energy metabolism to meet the needs of rapid growth of hindlimb thigh muscles by comparing to males in the same developmental period.(2)Through Pathway enrichment,the pathways related to the growth and adaption of unique life history in hindlimb thigh muscles were obtained,such as focal adhesion,tight junction,notch signaling pathway,insulin signaling pathway,m TOR signaling pathway and ECM-receptor interaction.These pathways varied between groups and played specific regulation effects at different developmental stages of the species.4.Construction of a non-reference RRBS method to assess DNA methylation levels in hindlimb thigh muscles at different developmental stages In this study,a RRBS method without reference species was successfully constructed to evaluate the DNA methylation level of hindlimb thigh muscles at different developmental stages in this species.The main type of DNA methylation in hindlimb thigh muscles of Chinese concave-eared frogs was CG.The methylation level in the upstream 2K region of the genes was significantly lower than that in the geneboby region.By pairwise comparison of 8 developmental stages,4836,4558,4781,4795,4745,5018 and 5494 DMRs were respectively obtained.GO enrichment of these genes mainly included developmental process,protein ubiquitination,focal adhesion,multicellular organism growth,amotor activity,etc.These items were different among different groups.5.Association analysis between differentially expressed genes and differential methylation-related genes in hindlimb thigh musclesThe results of correlation analysis of differentially expressed genes and differential methylation related genes showed that differentially expressed genes of negative correlation DMRs were 558,424,61,102,302,493,216 and 296 in 8comparison groups,respectively.The differentially expressed genes of DMRs in different comparison groups were enriched by Pathway,showing that some pathways were related to growth and development,including focal adhesion,tight junction,ECM receptor interaction and thyroid hormone synthesis.These pathways are different in different comparison groups,suggesting that DNA methylated modification could regulate the muscle growth and development of hindlimb thigh muscles.There were differentially expressed genes of DMRs related to muscle growth,development and movement in different comparison groups,such as TNNI2,MYOC and OBSCN,which further indicated that DNA methylated modification might directly regulate the growth and development of thigh muscle in hindlimb thigh muscles of this species.In addition,the differential level of methylation was also correlated with the differential expression of genes related to glucose metabolism,energy metabolism,protein synthesis and degradation,etc,such as,SDHAF2 related energy metabolism in G40(aquatic)and G42(terrestrial)comparison groups,NGB related to oxygen transport in pre-hibernation(3 months)and hibernation(5months)comparison groups,GCDH related to mitochondrial metabolism,PSMD8 related to protein degradation,PGM5 related to glycogen biosynthesis in the14-month-old male and female comparison groups,Eef1a2 related to translation extension factors of protein synthesis,KLHL40 related to protein degradation ubiquitin pathway,CD164 related to lysosomal pathway,etc.These results suggest that DNA methylated modification plays an important role in regulating the physiological metabolism of hindlimb thigh muscles growth,development and adaptation of the body to special life history stages.In this study,the development characters and molecular regulation mechanism of the hindlimb thigh muscles in the different life history of O.tormota were systematically studied and it lay a foundation for further study on the phenotypic differences between genders and their evolutionary adaptation. |
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