Intermuscular bones(IBs)are slender linear bones embedded in muscle,which ossify from tendons through a process of intramembranous ossification,and only exist in basal teleosts.IBs are essential for fish swimming,but ...Intermuscular bones(IBs)are slender linear bones embedded in muscle,which ossify from tendons through a process of intramembranous ossification,and only exist in basal teleosts.IBs are essential for fish swimming,but they present a choking risk during human consumption,especially in children,which can lead to commercial risks that have a negative impact on the aquaculture of these fish.In this review,we discuss the morphogenesis and functions of IBs,including their underlying molecular mechanisms,as well as the advantages and disadvantages of different methods for IB studies and techniques for breeding and generating IB-free fish lines.This review reveals that the many key genes involved in tendon development,osteoblast differentiation,and bone formation,e.g.,scxa,msxC,sost,twist,bmps,and osterix,also play roles in IB development.Thus,this paper provides useful information for the breeding of new fish strains without IBs via genome editing and artificial selection.展开更多
We documented the number, morphology, and distribution of intermuscular bones in five fishes of different ploidy： Carassius auratus （Abbr.WCC, 2n=100）, Carassius auratus variety PengZe （Abbr.PZCC, 3n=150）, improv...We documented the number, morphology, and distribution of intermuscular bones in five fishes of different ploidy： Carassius auratus （Abbr.WCC, 2n=100）, Carassius auratus variety PengZe （Abbr.PZCC, 3n=150）, improved triploid crucian carp （Abbr.ITCC, 3n=150）, improved red crucian carp （Carassius auratus red var., Abbr.IRCC, , 2n=100）, and improved allotet- raploids （Abbr.GAT, （, 4n=200）. The number of intermuscular bones in WCC, PZCC, and GxAT ranged from 78 to 83 （n =81）, 80 to 86 （n =84）, and 77 to 84 （n=82）, respectively. The numbers in ITCC and IRCC were significantly lower, ranging from 77 to 82 （ n =79） and 58 to 77 （ n =71）, respectively. The average number of intermuscular bones in each sarco- mere, ranked in order from highest to lowest, was 0.721 （WCC）, 0.673 （PZCC）, 0.653 （GAT）, 0.633 （ITCC）, and 0.608 （IRCC）. There was no difference between ITCC and GxAT or between GxAT and PZCC. However, the average number of intermuscular bones in the sarcomeres of ITCC, WCC, and PZCC differed significantly, as did that of IRCC and the four other kinds of fish. The intermuscular bone of these five fishes was divided into seven shape categories, non-forked （I）, one-end-unequal-bi-fork （）, one-end-equal-bi-fork （Y）, one-end-multi-fork, two-end-bi-fork, two-end-multi-fork, and tree- branch types. Generally, the morphological complexity was higher in the anterior intermuscular bones than in the posterior body. The number of intermuscular bones was similar but not equal between the left and right sides of the body. ITCC had sig- nificantly fewer intermuscular bones than either WCC or PZCC, making it of greater commercial value. Additionally, IRCC and ITCC had fewer intermuscular bones than WCC. Our observations are significant in both fish bone developmental biology and genetic breeding.展开更多
Intermuscular bones(IBs)are ossified from tendons and only occur in lower teleosts.Positive association between the regulation of scleraxis gene(scx)and tendon development gave us reasons to speculate that the scx gen...Intermuscular bones(IBs)are ossified from tendons and only occur in lower teleosts.Positive association between the regulation of scleraxis gene(scx)and tendon development gave us reasons to speculate that the scx gene may play a potential role in regulating the development of IBs.A phylogenetic analysis conducted for this study revealed potential functional differentiation between two scx orthologues,scxa and scxb.The scxa^(-/-) and scxb^(-/-) zebrafish were generated through CRISPR-Cas9 technology to study the role of scx in the IB and rib development.The results showed a significant reduction of the number of IBs in adult scxa-1^(-/-) zebrafish,with almost 70%reduction(15-25 IBs)compared to the wild type scxa-1^(+/+)zebrafish(76-80 IBs).In the scxa-1^(+/+)adults,IBs were observed in both dorsal and tail segments;however,in scxa-1^(-/-)fish IBs were observed only in the tail segment(none in the dorsal segment).Although scxa-1^(-/-) zebrafish had rib defects,the mutants were viable and fertile as adult fish.The scxb^(-/-) zebrafish had the same number of IBs and same skeletal phenotype as the wild-type fish.This suggests that only scxa has a crucial role in the IB development,and confirms functional differentiation of scx orthologues.To further clarify the molecular mechanism by which scxa affects the IB development,we conducted comparative transcriptome analysis of dorsal tissue samples of scxa-1^(+/+)(with IBs)and scxa-1^(-/-) (without IBs),and further verified the expression of key genes via qPCR.This is the first study to identify a gene that controls the amount of IBs in fish,and it provides a new sight into the effects of scxa on the molecular mechanism of IB development in fish.展开更多
为探究tent5c/fam46c基因(Family with sequence similarity 46c,家族序列相似性46 c)在斑马鱼(Danio rerio)肌间刺发育中的作用,选取11种鱼类分析tent5c系统演化特征,采用原位杂交技术和qRT-PCR分析tent5c基因在斑马鱼发育中的时空表...为探究tent5c/fam46c基因(Family with sequence similarity 46c,家族序列相似性46 c)在斑马鱼(Danio rerio)肌间刺发育中的作用,选取11种鱼类分析tent5c系统演化特征,采用原位杂交技术和qRT-PCR分析tent5c基因在斑马鱼发育中的时空表达特征,并利用CRISPR/Cas9建立了斑马鱼tent5c基因敲除纯合突变系(tent5c^(-/-)),对其骨骼表型和肌肉转录组进行分析。结果显示,tent5c编码TENT5C氨基酸在不同鱼类中具有高度的保守性,但在无肌间刺鱼类里发生了相同的氨基酸替换;tent5c基因在48hpf(Hours post fertilization)斑马鱼的眼睛和肌隔表达,在60dpf(Days post fertilization)斑马鱼的脊椎和肌间刺特异高表达。与野生型斑马鱼相比,tent5c^(-/-)斑马鱼肌间刺表现为生长减缓,矿化程度降低以及数量减少。比较转录组结果表明,在tent5c基因敲除之后,斑马鱼肌肉组织有112个基因表达下调,118个基因表达上调。差异基因主要富集于肌肉收缩和骨骼发育等相关通路,由此推测tent5c可能是通过调控这些肌肉和骨骼相关通路来对肌间刺发育产生影响。研究初步表明了tent5c基因在斑马鱼肌间刺发育中的作用,为进一步研究鱼类肌间刺等骨骼发育的分子调控机制提供了科学依据。展开更多
利用斑马鱼(Danio rerio)野生型与肌间刺完全缺失突变型个体,从骨骼染色和骨骼发育相关基因表达两方面,初步评价了肌间刺缺失对斑马鱼骨骼发育的影响。通过骨骼染色对比观察了两种肌间刺表型个体受精后8dpf(days post fertilization, d...利用斑马鱼(Danio rerio)野生型与肌间刺完全缺失突变型个体,从骨骼染色和骨骼发育相关基因表达两方面,初步评价了肌间刺缺失对斑马鱼骨骼发育的影响。通过骨骼染色对比观察了两种肌间刺表型个体受精后8dpf(days post fertilization, dpf)到56dpf的骨骼发育情况,结果显示,两种肌间刺表型除肌间刺外,其他骨骼发育基本同步。此外,通过qRT-PCR实验检测分析了6个骨骼发育相关基因(bmp2a、bmp4、smad1、smad4a、runx2a和sp7)在不同肌间刺表型5个胚胎发育时期(3hpf囊胚期、6hpf原肠胚期、12hpf体节期、24hpf咽囊期和72hpf孵化期)和5个胚后生长阶段(15、30、45、60和75dpf)的表达情况。结果显示:在胚胎发育时期,野生型和突变型个体中bmp2a、bmp4、smad1、smad4a基因和突变型个体中sp7基因的表达均呈现先升后降的变化趋势,且在体节期达到最高表达水平;野生型和突变型个体中runx2a基因和野生型个体中sp7基因则表现为逐渐上升的趋势。6个基因在囊胚期和原肠胚期表达量无显著差异, bmp2a的表达水平在体节期、咽囊期和孵化期无显著差异,野生型个体bmp4、smad1、smad4a、runx2a基因在体节期、咽囊期和孵化期的表达水平明显高于突变型,而sp7基因则表现为突变型明显高于野生型。胚后发育阶段6个基因在5个生长阶段均呈现逐渐下降的趋势,且在两种肌间刺表型间其表达仅在个别时期差异显著。综上所述,肌间刺的缺失对斑马鱼骨骼发育表现型无显著影响,只在胚胎发育时期影响骨骼相关基因表达水平的变化;结合骨骼染色结果,推测肌间刺缺失对斑马鱼骨骼发育无显著影响。展开更多
基金This work was supported by the National Natural Science Foundation of China(U1702233 and U1902202)Program of the Chinese Academy of Sciences(XDA24030505 and XDA23080502)Program of Yunnan Provincial Science and Technology Department(202003AD150017 and 2018FY001-007)。
文摘Intermuscular bones(IBs)are slender linear bones embedded in muscle,which ossify from tendons through a process of intramembranous ossification,and only exist in basal teleosts.IBs are essential for fish swimming,but they present a choking risk during human consumption,especially in children,which can lead to commercial risks that have a negative impact on the aquaculture of these fish.In this review,we discuss the morphogenesis and functions of IBs,including their underlying molecular mechanisms,as well as the advantages and disadvantages of different methods for IB studies and techniques for breeding and generating IB-free fish lines.This review reveals that the many key genes involved in tendon development,osteoblast differentiation,and bone formation,e.g.,scxa,msxC,sost,twist,bmps,and osterix,also play roles in IB development.Thus,this paper provides useful information for the breeding of new fish strains without IBs via genome editing and artificial selection.
基金supported by Special Fund for Agro-scientific Research in the Public Interest (200903046)the National Natural Science Foundation of China (30930071)+5 种基金Major International Cooperation Projects of the National Natural Science Foundation of China (31210103918)Innovative Research Groups of Hunan Provincial Natural Science Fund (10JJ7004)the National High Technology Research and Development Program of China (2011AA100403)Pre-project of the National Key Basic Research and Development Program (2012CB722305)the Construct Program of the Key Discipline in Hunan ProvinceConstruct Program of the National Key Discipline
文摘We documented the number, morphology, and distribution of intermuscular bones in five fishes of different ploidy： Carassius auratus （Abbr.WCC, 2n=100）, Carassius auratus variety PengZe （Abbr.PZCC, 3n=150）, improved triploid crucian carp （Abbr.ITCC, 3n=150）, improved red crucian carp （Carassius auratus red var., Abbr.IRCC, , 2n=100）, and improved allotet- raploids （Abbr.GAT, （, 4n=200）. The number of intermuscular bones in WCC, PZCC, and GxAT ranged from 78 to 83 （n =81）, 80 to 86 （n =84）, and 77 to 84 （n=82）, respectively. The numbers in ITCC and IRCC were significantly lower, ranging from 77 to 82 （ n =79） and 58 to 77 （ n =71）, respectively. The average number of intermuscular bones in each sarco- mere, ranked in order from highest to lowest, was 0.721 （WCC）, 0.673 （PZCC）, 0.653 （GAT）, 0.633 （ITCC）, and 0.608 （IRCC）. There was no difference between ITCC and GxAT or between GxAT and PZCC. However, the average number of intermuscular bones in the sarcomeres of ITCC, WCC, and PZCC differed significantly, as did that of IRCC and the four other kinds of fish. The intermuscular bone of these five fishes was divided into seven shape categories, non-forked （I）, one-end-unequal-bi-fork （）, one-end-equal-bi-fork （Y）, one-end-multi-fork, two-end-bi-fork, two-end-multi-fork, and tree- branch types. Generally, the morphological complexity was higher in the anterior intermuscular bones than in the posterior body. The number of intermuscular bones was similar but not equal between the left and right sides of the body. ITCC had sig- nificantly fewer intermuscular bones than either WCC or PZCC, making it of greater commercial value. Additionally, IRCC and ITCC had fewer intermuscular bones than WCC. Our observations are significant in both fish bone developmental biology and genetic breeding.
基金This work was financially supported by the National Natural Science Foundation of China(Grant No.31872559)National Key Research and Development Program(Grant No.2018YFD0900102)+2 种基金Modern Agriculture Industry Technology System Construction Projects of China titled as-Staple Freshwater Fishes Industry Technology System(Grant No.CARS-46-08)Fundamental Research Funds for the Central Universities(Grant No.2662018PY035)State Key Laboratory of Developmental Biology of Freshwater Fish(2018KF003).
文摘Intermuscular bones(IBs)are ossified from tendons and only occur in lower teleosts.Positive association between the regulation of scleraxis gene(scx)and tendon development gave us reasons to speculate that the scx gene may play a potential role in regulating the development of IBs.A phylogenetic analysis conducted for this study revealed potential functional differentiation between two scx orthologues,scxa and scxb.The scxa^(-/-) and scxb^(-/-) zebrafish were generated through CRISPR-Cas9 technology to study the role of scx in the IB and rib development.The results showed a significant reduction of the number of IBs in adult scxa-1^(-/-) zebrafish,with almost 70%reduction(15-25 IBs)compared to the wild type scxa-1^(+/+)zebrafish(76-80 IBs).In the scxa-1^(+/+)adults,IBs were observed in both dorsal and tail segments;however,in scxa-1^(-/-)fish IBs were observed only in the tail segment(none in the dorsal segment).Although scxa-1^(-/-) zebrafish had rib defects,the mutants were viable and fertile as adult fish.The scxb^(-/-) zebrafish had the same number of IBs and same skeletal phenotype as the wild-type fish.This suggests that only scxa has a crucial role in the IB development,and confirms functional differentiation of scx orthologues.To further clarify the molecular mechanism by which scxa affects the IB development,we conducted comparative transcriptome analysis of dorsal tissue samples of scxa-1^(+/+)(with IBs)and scxa-1^(-/-) (without IBs),and further verified the expression of key genes via qPCR.This is the first study to identify a gene that controls the amount of IBs in fish,and it provides a new sight into the effects of scxa on the molecular mechanism of IB development in fish.
文摘为探究tent5c/fam46c基因(Family with sequence similarity 46c,家族序列相似性46 c)在斑马鱼(Danio rerio)肌间刺发育中的作用,选取11种鱼类分析tent5c系统演化特征,采用原位杂交技术和qRT-PCR分析tent5c基因在斑马鱼发育中的时空表达特征,并利用CRISPR/Cas9建立了斑马鱼tent5c基因敲除纯合突变系(tent5c^(-/-)),对其骨骼表型和肌肉转录组进行分析。结果显示,tent5c编码TENT5C氨基酸在不同鱼类中具有高度的保守性,但在无肌间刺鱼类里发生了相同的氨基酸替换;tent5c基因在48hpf(Hours post fertilization)斑马鱼的眼睛和肌隔表达,在60dpf(Days post fertilization)斑马鱼的脊椎和肌间刺特异高表达。与野生型斑马鱼相比,tent5c^(-/-)斑马鱼肌间刺表现为生长减缓,矿化程度降低以及数量减少。比较转录组结果表明,在tent5c基因敲除之后,斑马鱼肌肉组织有112个基因表达下调,118个基因表达上调。差异基因主要富集于肌肉收缩和骨骼发育等相关通路,由此推测tent5c可能是通过调控这些肌肉和骨骼相关通路来对肌间刺发育产生影响。研究初步表明了tent5c基因在斑马鱼肌间刺发育中的作用,为进一步研究鱼类肌间刺等骨骼发育的分子调控机制提供了科学依据。
