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Single-cell transcriptomes and runx2b-/- mutants reveal the genetic signatures of intermuscular bone formation in zebrafish

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National Science Review 2022年第11期9卷 97-107页

作者： Chun-Hong Nie Shi-Ming Wan Yu-Long Chen Ann Huysseune Ya-Ming Wu Jia-Jia Zhou Alexandre Wagner Silva Hilsdorf Wei-Min Wang Paul Eckhard Witten Qiang Lin Ze-Xia Gao College of Fisheries Key Lab of Freshwater Animal BreedingMinistry of Agriculture and Rural AffairsKey Lab of Agricultural Animal GeneticsBreeding and Reproduction of Ministry of EducationEngineering Research Center of Green Development for Conventional Aquatic Biological Industry in the Yangtze River Economic Belt Ministry of Education Huazhong Agricultural University Hubei Hongshan Laboratory CAS Key Laboratory of Tropical Marine Bio-Resources and Ecology South China Sea Institute of Oceanology Innovation Academy of South China Sea Ecology and Environmental Engineering Chinese Academy of Sciences Department of Biology Ghent University Unit of Biotechnology University of Mogi das Cruzes Guangdong Laboratory for Lingnan Modern Agriculture

Intermuscular bones（Ibs） are mineralized spicules, present in the myosepta of many, but not all, teleost species. Ibs are often small and sharp, and they consequently limit how the fish can be processed; the Ibs may cause injury or trauma if lodged in consumers' throats or mouths, and therefore affect the appeal of the fish to many consumers. The development of Ibs in teleosts is still not fully understood and the molecular basis of Ib development remains to be established. Here, the characteristics of Ib tissue are evaluated based on single-cell transcriptomics in wild-type zebrafish. The analysis defined 18 distinct cell *** trajectories showed that Ibs are derived from tendons and that a core tendon-osteoblast cell lineage is related to Ib formation. In particular, the functions of 10 candidate genes were evaluated via CRISPR-Cas9 mutants. Among those, runx2b-/-mutants completely lost Ibs, while swimming performance, growth and bone mineral density were not significantly different from runx2b+/+*** single-cell RNA sequencing（sc RNA-seq） analysis in runx2b-/-and runx2b+/+zebrafish revealed the role of osteoblasts in Ib formation. In addition, differentially expressed genes were enriched in the transforming growth factor β/bone morphogenetic protein（TGF-β/bMP） pathway after runx2b deletion. This study provides evidence for the crucial role of runx2b regulation in Ib formation. Genetic breeding can target runx2b regulation and generate strains of commercial fish species without Ibs, which can improve the safe consumption and economic value of many farmed fish species.

关键词： teleosts zebrafish intermuscular bones scRNA-seq tendon-osteoblast cell lineage runx2b genetic breeding

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Effect of runx2b deficiency in intermuscular bones on the regulatory network of lncRNA-miRNA-mRNA

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COMPARATIVE bIOCHEMISTRY AND PHYSIOLOGY D-GENOMICS & PROTEOMICS 2024年 49卷 101171页

作者： Xiao, Zhengyu Chen, Yulong Wang, Xudong Sun, Qiujie Tu, Tan Liu, Junqi Nie, Chunhong Gao, Zexia Huazhong Agr Univ Coll FisheriesKey Lab Agr Anim Genet Breeding & R Minist Agr & Rural AffairsMinist Educ Key Lab Freshwater Anim BreedingEngn Res Ctr Gree Wuhan 430070 Peoples R China Hubei Hongshan Lab Wuhan 430070 Peoples R China

Intermuscular bones (Ibs) are mineralized spicules that negatively impact the quality and value of fish products. runx2b is a crucial modulator in promoting bone formation through regulating osteoblast differentiation. Pre-vious studies suggested that loss of runx2b gene completely inhibited Ibs formation in zebrafish. However, how the whole transcriptome, including mRNA and non-coding RNA (ncRNA), affects the Ibs development in run- x2b-/-zebrafish are not known. The aim of this study was to identify the regulatory networks of differentially expressed (DE) lncRNAs, miRNAs, and mRNAs in zebrafish with and without Ibs (runx2b+/+ fish and runx2b-/-fish) utilizing high-throughput sequencing techniques. All together there are 1051 mRNAs, 456 lncRNAs, and 18 miRNAs differentially expressed were found between these two strains. The analysis of Kyoto Encyclopedia of Genes and Genomes (KEGG) has highlighted significant pathways linked to the development of Ibs, specifically the TGF-beta and Wnt signaling pathways, and a number of genes concentrated on these two signaling pathways related to the formation of Ibs. Further, 1989 competing endogenous RNA (ceRNA) networks were created ac-cording to the correlation among mRNAs, miRNAs and lncRNAs. The ceRNA networks results revealed 52 ceRNA pairs related to the Ibs formation, consisting of 52 mRNAs, 37 lncRNAs, and 6 miRNAs. Of these, we found that dre-miR-2189 was the key element of ceRNA pairs, interacting with 19 mRNAs and 11 lncRNAs, and MSTRG.13175.1 could regulate sp7 expression by interacting with dre-miR-2189 to function in osteogenic dif-ferentiation. Subsequent experiments at the cellular level also revealed the interaction mechanism. The outcomes indicated a crucial role of miRNAs and lncRNAs in the development of fish Ibs, which offer new views into the functions of ncRNAs involved in Ibs formation.

关键词： Fish Intermuscular bones runx2b LncRNAs and miRNAs CeRNA

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Generation of blunt snout bream without intermuscular bones by runx2b gene mutation

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AQUACULTURE 2023年 567卷

作者： Dong, Qiang Nie, Chun-Hong Wu, Ya-Ming Zhang, Dong-Yang Wang, Xu-Dong Tu, Tan Jin, Jian Tian, Zu-Yu Liu, Jun-Qi Xiao, Zheng-Yu Wan, Shi-Ming Gao, Ze-Xia Huazhong Agr Univ Coll Fisheries Engn Res Ctr Green Dev Convent Aquat Biol Ind Yang Minist Agr & Rural AffairsMinist EducEngn Techno Wuhan 430070 Peoples R China Guangdong Lab Lingnan Modern Agr Guangzhou 510000 Peoples R China Huazhong Agr Univ Coll Fisheries Wuhan 430070 Hubei Peoples R China

blunt snout bream (Megalobrama amblycephala) is an important herbivorous freshwater economic fish cultivated in China. Its economic value has been affected by the existence of intermuscular bones (Ibs). Our previous study proved that runx2b is the key gene for the Ibs formation, and lack of runx2b gene could obtain a stable zebrafish mutant without Ibs. We found runx2b mutant F0 generation of blunt snout bream through CRISPR/Cas9 editing technology had a significant reduction on the number of Ibs. In order to further obtain the blunt snout bream without Ibs, we used the runx2b mutant F0 generation to self-bred to obtain F1 generation. Subsequently, the 35 runx2b mutant F1 individuals of blunt snout bream without Ibs were genotyped and found the number of runx2b targeting sites 1, 2 and 3 obtained 3 (1 insertion, 2 deletion), 13 (8 insertion, 5 deletion) and 7 (1 insertion, 6 deletion) mutation types with the mutation efficiency being 25.71%, 94.28%, and 71.43%, respectively. Alizarin red bone staining and X-ray scan analysis showed that Ibs were not existed in runx2b mutant F1 individuals, and micro-CT analysis found that the mineralization of other bone in runx2b mutant was not significantly affected compared to wild type fish (P > 0.05). RT-qPCR results also exhibited that bone-related genes had a lower expression level in runx2b mutants compared to wild type fish. Subsequently, we used the national standard methods to detect nutrition composition of muscle tissue in wild type (with Ibs) and runx2b mutant F1 individuals (without Ibs) at 120 dpf. The results showed that the basic muscle nutritional composition (moisture, crude protein, crude lipid and crude ash), and the contents of most amino acids and fatty acids had no significant differences between wild and runx2b mutant at juvenile stage (P > 0.05). This study proves that the runx2b gene has an essential role for Ibs formation in aquaculture species and lays an important foundation for obtaining a new blunt s

关键词： Megalobrama amblycephala Intermuscular bones runx2b Genotype Phenotype

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Creation of intermuscular bone-free mutants in amphitriploid gibel carp by editing two duplicated runx2b homeologs

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AQUACULTURE 2023年 567卷

作者： Gan, Rui-Hai Li, Zhi Wang, Zhong-Wei Li, Xi-Yin Wang, Yang Zhang, Xiao-Juan Tong, Jin-Feng Wu, Yue Xia, Ling-Yun Gao, Ze-Xia Zhou, Li Gui, Jian-Fang Chinese Acad Sci Inst Hydrobiol Innovat Acad Seed Design State Key Lab Freshwater Ecol & BiotechnolHubei H Wuhan 430072 Peoples R China Univ Chinese Acad Sci Beijing 100049 Peoples R China Huazhong Agr Univ Coll Fisheries Engn Res Ctr Green Dev Convent Aquat Biol Ind Yang Hubei Hongshan LabKey Lab Freshwater Anim Breedin Wuhan 430070 Peoples R China Chinese Acad Sci Inst Hydrobiol 7 Donghu South Rd Wuhan 430072 Hubei Peoples R China Huazhong Agr Univ Coll Fisheries Wuhan 430070 Hubei Peoples R China

Intermuscular bones (Ibs) are ubiquitous in most of freshwater farmed fishes, especially in cyprinid species, and the creation of Ib-free mutants has become a hot issue in fish genetic breeding because of the potential beneficial for consumers and fish processing industry. Recently, significant progress in the mechanism underlying Ib development has been achieved in zebrafish and a decisive player, runt-related transcription factor 2b (runx2b), has been identified. However, its role has not yet been validated in other farmed fishes. Amphitriploid gibel carp (Carassius gibelio) is an important aquaculture species in China, but the injuries threats with approximately 80 Ibs have strongly restricted its industry development. In this study, we first identified two homeologs of runx2b (Cgrunx2b-A and Cgrunx2b-b) in gibel carp with about 91.8% identity, each of them possessing three alleles with 99.6% identities. Then, we traced the Ib ossification process. Alizarin red S staining showed that gibel carp Ib began to ossify at 14 approximately days post hatching (dph) and developed from the posterior to the anterior regions. Cgrunx2b-A and Cgrunx2b-b displayed similar expression pattern but differential expression level during Ib development. Moreover, we applied an optimized protocol of CRISPR/Cas9 editing in the polyploid Carassius complex to disrupt the functions of Cgrunx2b-A and Cgrunx2b-b singly or simultaneously. Ib development was unaffected by single deficiency of Cgrunx2b-A or Cgrunx2b-b, but simultaneous disruption of all alleles of both Cgrunx2b-A and Cgrunx2b-b leads to complete loss of Ib. The results indicate that Cgrunx2b-A and Cgrunx2b-b cooperatively regulate Ib development. Importantly, we created several gynogenetic gibel carp mutants, including Ib-free Cgrunx2b-A / /;Cgrunx2b-b / / mutants and Ib-less Cgrunx2b-A / /;Cgrunx2b-b / /+/- mutants. The current study not only confirms the cooperatively decisive role of two homeologs of runx2b in Ib formation,

关键词： Intermuscular bone runx2b Gibel carp Gynogenesis Homeolog

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翘嘴鲌runx2b基因的克隆与表达特征分析

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浙江农业学报 2024年第5期36卷 1024-1031页

作者：何昌熙郑建波马建波贾永义刘士力蒋文枰迟美丽程顺李飞上海海洋大学水产与生命学院农业农村部淡水水产种质资源重点实验室上海201306 浙江省淡水水产研究所浙江省淡水水产遗传育种重点实验室浙江湖州313001 余姚市水产技术推广中心浙江余姚315400

为探究runx2b基因在翘嘴鲌肌间刺骨化过程中的调控作用,基于转录组数据和PCR扩增技术克隆获得了翘嘴鲌runx2b基因全长编码序列。通过茜素红整体骨骼染色对肌间刺(Ibs)形成的不同阶段进行了分期,同时采用qRT-PCR技术检测了runx2b基因的... 详细信息

为探究runx2b基因在翘嘴鲌肌间刺骨化过程中的调控作用,基于转录组数据和PCR扩增技术克隆获得了翘嘴鲌runx2b基因全长编码序列。通过茜素红整体骨骼染色对肌间刺(Ibs)形成的不同阶段进行了分期,同时采用qRT-PCR技术检测了runx2b基因的时空表达水平。结果显示,runx2b基因开放阅读框为1 401 bp,编码466个氨基酸,预测分子量为50.676 ku,理论等电点为9.35;氨基酸序列多重比对显示,翘嘴鲌runx2b氨基酸序列与近源物种的同源性高达95%以上,且都具有高度保守的Runt和runxI结构域;系统进化树分析表明,翘嘴鲌与团头鲂的亲缘关系最为接近。茜素红染色结果显示,在肌间刺发育前,翘嘴鲌的主轴骨骼和附肢骨骼已经发育完全;其中,孵化后15 d,尾部肌隔中开始出现小肌间刺;孵化后30 d,翘嘴鲌幼体的肌间刺发育完全。组织表达结果显示,runx2b基因在翘嘴鲌各组织中均有表达,鳃中的表达量最高,其次是肌肉。此外,runx2b基因在翘嘴鲌幼体不同生长阶段的表达模式与肌间刺骨化发生的时序保持一致,提示该基因与肌间刺骨化的相关性。研究结果可以为后续无肌间刺翘嘴鲌新种质的创制和肌间刺形成的分子机制提供基础数据。

关键词：翘嘴鲌肌间刺 runx2b 基因表达骨化

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基于runx2b基因敲除的无肌间刺团头鲂新种质创制

基于runx2b基因敲除的无肌间刺团头鲂新种质创制

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作者：董强华中农业大学

学位级别：硕士

我国主要养殖鱼类均存在一定数量的肌间刺,一定程度上影响了其加工和食用价值,还有受伤害的风险。近年来,肌间刺性状已成为鱼类遗传育种研究领域备受关注的改良性状之一。已有研究证明runx2b是鱼类肌间刺形成的关键基因,runx2b基因缺失... 详细信息

我国主要养殖鱼类均存在一定数量的肌间刺,一定程度上影响了其加工和食用价值,还有受伤害的风险。近年来,肌间刺性状已成为鱼类遗传育种研究领域备受关注的改良性状之一。已有研究证明runx2b是鱼类肌间刺形成的关键基因,runx2b基因缺失可以获得稳定的无肌间刺斑马鱼突变体,但尚未有研究聚焦于该基因的无肌间刺经济鱼类的研究。本研究以经济型鱼类团头鲂(Megalobrama amblycephala)为主要研究对象,利用CRISPR-Cas9基因编辑技术,构建缺失runx2b功能的突变体F0代,并筛选出肌间刺减少30%以上的突变体F0代,待其发育至性成熟进行自交繁殖,获得F1代;当F1代的群体生长至四月龄时,通过X光活体扫描F1代个体,筛选无肌间刺团头鲂,随后通过阳性克隆技术检测无肌间刺团头鲂F1代的基因型;采用Micro-CT分析野生型和无肌间刺团头鲂F1代骨骼的骨密度;茜素红整体骨骼染色法和X光扫描技术观察野生型和无肌间刺团头鲂F1代的骨骼发育情况;进一步采用q RT-PCR技术分析骨发育相关基因在野生型和无肌间刺团头鲂F1代尾部肌肉组织中的表达模式,并对野生型(含肌间刺)和团头鲂F1代(无肌间刺)肌肉的营养成分进行了分析。主要研究结果如下:1.团头鲂runx2b突变体F0代的构建、表型及基因表达模式分析本研究通过CRISPR-Cas9基因编辑技术,在团头鲂受精卵的1细胞期之前进行多靶点注射,构建runx2b突变体F0代。采用2%琼脂糖凝胶电泳法检测48h受精卵的突变情况,检测结果为双条带的个体,进一步采用Sanger测序法测序并进行序列比对,靶点附近出现双峰则证明敲除成功;随机选取54尾70dpf的已筛选出的runx2b突变体F0代进行茜素红骨骼染色。结果显示,其中有6尾表现为肌间刺部分缺失的性状,肌间刺缺失的数量分别为43根、7根、17根、14根、7根、14根,肌间刺缺失比率分别为33.86%、5.74%、13.60%、11.48%、5.60%和11.29%。与野生型相比,少刺突变体F0代在背部和尾部均出现肌间刺部分缺失的情况,且脊椎、肋骨等其他骨骼发育正常;采用q RT-PCR技术分析发育到3月龄的runx2b突变体F0代尾部肌肉中骨发育相关基因的表达情况。结果显示,由于团头鲂突变体F0代中runx2b基因的缺失,与矿化相关的基因(entpd5a、entpd5b、spp1、scpp1)在runx2b突变体F0代中的表达量与野生型相比均显著降低(P<0.05),而与肌腱发育相关基因tnmd在runx2b突变体F0代中的表达量与野生型相比没有显著差异(P>0.05)。2.无肌间刺团头鲂突变体F1代的培育、表型及基因表达模式分析为获得无肌间刺的团头鲂突变体F1代,本实验以筛选获得的团头鲂runx2b突变体F0代为基础,选择性成熟、肌间刺减少30%以上的runx2b突变体F0代进行强化培育,挑选发育良好的runx2b突变体F0代作为F1代团头鲂的亲本,自交繁殖获得F1代;随后选择60尾45dpf的runx2b突变体F1代进行茜素红骨骼染色观察表型。结果显示,27尾(占45%)runx2b突变体F1代的肌间刺完全缺失,且其他骨矿化正常,如肋骨、脊椎、神经棘和血管棘。当团头鲂runx2b突变体F1代生长至四月龄时,使用X射线对runx2b突变体F1代进行活体扫描,并从5000尾中筛选出共316尾完全没有肌间刺的团头鲂,Micro-CT结果也进一步明确,runx2b突变体F1代完全没有肌间刺。组织骨密度分析结果显示,生长至4月龄的野生型和runx2b突变体F1代团头鲂的脊椎、肋骨、神经棘、血管棘等骨骼的组织骨密度(tissue mineral density,TMD)值没有显著差异(P>0.05);为探究runx2b基因功能缺失的团头鲂突变体F1代的基因表达模式,随机选取发育到4月龄的runx2b突变体F0代,采用q RT-PCR技术分析其尾部肌肉中骨发育相关基因的表达情况。结果显示,由于无肌间刺团头鲂F1代中runx2b基因的缺失,与骨矿化相关的基因(alpl、bglap、entpd5a、entpd5b)在无肌间刺团头鲂F1代中的表达量与野生型相比均显著降低,且成骨相关基因sp7在无肌间刺团头鲂F1代中的表达量与野生型相比也显示出相对较低的表达水平。3.无肌间刺团头鲂突变体F1代的基因型分析和肌肉的营养成分分析为确定无肌间刺团头鲂突变体F1代的runx2b基因型,随机选择35尾无肌间刺团头鲂F1代进行阳性克隆检测。结果显示,runx2b靶点1、2、3突变类型分别为:靶点1为3种突变类型(1种插入类型,2种缺失类型),插入2 bp,缺失5 bp、8 bp;靶点2为13种突变类型(8种插入,5种缺失类型),插入4bp、5 bp、6 bp、8

关键词：肌间刺团头鲂 runx2b 基因敲除表型分析基因分型差异表达基因分析营养成分分析

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Zebrafish furin mutants reveal intricacies in regulating Endothelin1 signaling in craniofacial patterning

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DEVELOPMENTAL bIOLOGY 2006年第1期295卷 194-205页

作者： Walker, Macie b. Miller, Craig T. Talbot, Jared Coffin Stock, David W. Kimmel, Charles b. 1254 Univ Oregon Inst Neurosci Eugene OR 97403 USA Univ Colorado Dept Ecol & Evolut Biol Boulder CO 80309 USA

Endothelin1 (Edn1) signaling promotes ventral character to the facial skeleton. In zebrafish edn1 mutants, the ventral jaw structures are severely reduced and fused to their dorsal counterparts, with a loss of joints that normally form at an intermediate dorsal-ventral position. Loss of function at another locus, sturgeon, also yields joint losses, but only mild reductions in the ventral jaw structures. We show that sturgeon encodes one of two orthologs of Furin present in zebrafish, and that both furin genes may function partially redundantly to activate Edn1 signaling. Supporting this hypothesis, early expression of edn1-dependent genes is downregulated in sturgeon (furinA) mutants. Later in development, expression of most of these genes recovers to near wild-type levels in furinA mutants but not in edn1 mutants. The recovery explains the less severe furinA mutant skeletal phenotype and suggests that late gene expression is dependent on a critical level of Edn1 signaling not present in the more severe edn1 mutants. However, expression defects in the intermediate joint-forming domains in both mutants persist, explaining the joint losses observed later in both mutants. We further show that in both mutants the arches fail to correctly undergo ventral elongation before skeletogenesis begins and propose a model in which this failure is largely responsible for the loss of an Edn1-dependent compartmentation of the arch into the intermediate and ventral domains. (c) 2006 Elsevier Inc. All rights reserved.

关键词： sturgeon form endothelin1 Dlx barx1 Sox9a runx2b

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外源p204和cbfa1对斑马鱼胚胎发育和骨生成的影响

外源p204和cbfa1对斑马鱼胚胎发育和骨生成的影响

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作者：罗在礼山东大学

学位级别：硕士

骨骼有两个明显的特征：一是广泛分布于躯体并且组成骨骼的元件形状形态具有独特性,二是骨骼由两种不同的组织(成骨和软骨)组成。骨骼的形成主要通过软骨内成骨(endochondral ossification)和膜内成骨(intramembranous ossification)两... 详细信息

骨骼有两个明显的特征：一是广泛分布于躯体并且组成骨骼的元件形状形态具有独特性,二是骨骼由两种不同的组织(成骨和软骨)组成。骨骼的形成主要通过软骨内成骨(endochondral ossification)和膜内成骨(intramembranous ossification)两种方式。脊椎动物骨骼的生成是一个复杂并高度有序的过程。作为转录生长因子runt/Cbfa家族的一个成员,Cbfal(runx2)通过其runt结构域结合到DNA上激活成骨细胞中骨钙蛋白基因的表达。在成骨分化过程中,p204作为Cbfa1的辅助激活因子共同促进成骨细胞的分化。bMP2诱导间充质细胞C2C12向成骨细胞分化过程中,p204表达量增加；p204,Cbfa1和pRb蛋白组成三元复合体共同参与了成骨细胞分化的过程。模式动物斑马鱼因其个体小、性成熟周期短、体外受精和发育、胚体透明容易观察、便于显微操作等优势,现已用于发育生物学相关研究,取得了许多进展。目前,虽然斑马鱼还没有广泛应用于骨发育和相关疾病的研究,但已有许多研究证明了其可行性。斑马鱼骨骼可以分头部骨骼、躯体骨骼和鳍三种,在骨骼生成与发育过程中有着从头到尾的有序骨化的过程,头部首先形成,接着躯干骨化,与躯干骨化的同时鳍逐渐形成。Manuel等认为斑马鱼的鳍质鳞条虽然不是一个典型的骨骼组织,但其再生过程对于骨组织功能紊乱的临床前研究具有重要的意义。我们以斑马鱼为模式动物,采用显微注射的方式将外源p204和cbfa1导入其单细胞胚胎内,通过观察胚胎中融合蛋白GFP的荧光表达,发现两种融合质粒在斑马鱼胚胎中从8 hpf时期开始表达,表达位置主要集中在胚胎的胸腹部,头部和尾鳍处有较少表达。胚胎发育至8 dpf时,荧光表达量开始减少,约12 dpf后荧光彻底消失。两种质粒p204-EGFP和cbfal-EGFP注射诱导斑马鱼幼鱼产生大量畸形,以腹部膨大、脊椎弯曲、心胸部变异、尾部弯曲变短、头部畸形等为主。这些较严重畸形幼鱼只有少部分可存活下去。通过qPCR检测发现外源p204和cbfa1单独注射都可以诱导斑马鱼骨骼生成相关标志基因和关键基因osx, vent,tcf7表达量显著上调,而p204和cbfa1双质粒注射组相关基因的表达量高于任一单独注射组。进一步通过原位杂交检测OSX,tcf7在质粒注射后的时空表达,研究结果表明这两个基因表达时间提前,并且表达区域也有所扩展。同时,外源p204和cbfa1都可以诱导促进斑马鱼软骨,特别是pharyngeal arches软骨的分化,刺激成骨的形成,与相关成骨基因表达量高于单独注射组结果类似,在促进骨骼的生长和软骨的分化过程中,双质粒注射组结果也更加明显。研究表明：1.外源p204和cbfa1都可以参与斑马鱼骨骼生成分化过程中,并在一定程度上促进或更改(造成畸形发育)这一进程：2.p204可以促进cbfa1(或runx2b)作为骨骼生长中心因子参与骨骼生成,二者共同作用促进斑马鱼硬骨形成。本研究为进一步以斑马鱼为模式动物阐明以runx2b为中心的骨骼发生相关的分子机制提供了依据。

关键词：斑马鱼骨骼发生 p2O4 cbfa1 runx2b

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p204注射对斑马鱼胚胎发育和早期骨形成的影响

p204注射对斑马鱼胚胎发育和早期骨形成的影响

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作者：赵海滨山东大学

学位级别：硕士

哺乳动物骨骼的发育经过了多步过程,包括早期骨的图式形成、间充质细胞分化为成骨细胞系和软骨细胞系、造血干细胞分化为破骨细胞系,以及前体细胞终末分化为三种类型的细胞,即软骨细胞、成骨细胞和破骨细胞。成骨细胞的发生是一个非常... 详细信息

哺乳动物骨骼的发育经过了多步过程,包括早期骨的图式形成、间充质细胞分化为成骨细胞系和软骨细胞系、造血干细胞分化为破骨细胞系,以及前体细胞终末分化为三种类型的细胞,即软骨细胞、成骨细胞和破骨细胞。成骨细胞的发生是一个非常复杂的过程,与多种信号通路相关,如TGF-p信号通路、Wnt信号通路、FGF信号通路、Hedgehog信号通路等。在这些信号通路中,转录因子Cbfa1/runx2(core binding factor a 1)起关键作用,是成骨细胞分化和发生最重要的转录因子。 p204是小鼠干扰素诱导p200蛋白家族的成员之一,这一蛋白家族的成员通过与靶蛋白(包括转录因子,信号蛋白,肿瘤蛋白和肿瘤抑制因子)的结合来调节其活性,在细胞增殖和组织分化中起着重要的作用。已有研究表明,p204作为Cbfal蛋白的辅因子,以pRb为桥梁直接或间接结合到Cbfa1上促进骨的生长；p204、pRb与Cbfa1构成一个三元转录复合体促进Cbfa1下游骨发生相关基因(如ALP, OCL)的转录。通过基因序列比对分析表明,在斑马鱼中存在runx2, pRb的同源基因,其中runx2的同源基因有两个,分别为runx2a和runx2b; runx2b在斑马鱼骨发生中起主要作用。已有研究表明,runx2b是一种母原型的基因,在1-cell期利用MO抑制其表达会导致斑马鱼胚胎的严重背部化。但未找到p204的同源基因,本课题以斑马鱼为模式动物主要研究外源基因p204在胚胎发育以及早期骨发生过程中的作用。实验方法：(一)构建p204与GFP的融合蛋白表达载体。(二)将构建好的表达载体直接显微注射到斑马鱼1-cell胚胎中。(三)检测p204注射后对斑马鱼胚胎发育和早期骨形成的影响。1)在倒置显微镜下观察GFP的表达情况以指示p204基因的表达。2)通过real-time RT-PCR来检测几种斑马鱼背腹轴形成和骨分化相关基因表达水平的变化。3)原位杂交技术检测p204注射后runx2b的表达变化。研究结果：1)p204注射诱导产生斑马鱼腹部膨大和脊柱弯曲的畸形。2)GFP观察结果表明,p204在斑马鱼胚胎发育的8hpf-8dpf均有大量的表达,但在11dpf之后表达几乎完全消失。3)real-time RT-PCR结果表明,在1dpf-3dpf,一些与斑马鱼背腹轴形成和骨分化相关的基因如vent, runx2b, osn等在注射前后表达水平发生变化。4)runx2b原位杂交结果显示,在p204注射后unx2b的表达量有明显上调,但其表达的空间位置变化不大。结论：p204注射到斑马鱼胚胎后,可诱导产生斑马鱼腹部膨大的畸形,并引起了一些与斑马鱼背腹轴形成和骨分化相关的基因如vent, runx2b, osn等表达水平发生变化。本研究不仅发现了p204对斑马鱼胚胎发育的作用,而且为进一步阐明斑马鱼骨发生相关的分子机制提供了依据。

关键词： p204 胚胎发育 runx2b osn 斑马鱼

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草鱼肌间刺发生发育特征及无肌间刺草鱼新种质培育

草鱼肌间刺发生发育特征及无肌间刺草鱼新种质培育

引用

作者：王正银华中农业大学

学位级别：硕士

肌间刺性状的遗传改良已成为鱼类遗传育种研究的重要方向之一。草鱼（Ctenopharyngodon idella）作为我国大宗淡水经济鱼类的重要养殖对象,其养殖产量常年稳居全国首位,是世界上产量最高的淡水经济鱼类。草鱼养殖产业已成为我国淡水鱼... 详细信息

肌间刺性状的遗传改良已成为鱼类遗传育种研究的重要方向之一。草鱼（Ctenopharyngodon idella）作为我国大宗淡水经济鱼类的重要养殖对象,其养殖产量常年稳居全国首位,是世界上产量最高的淡水经济鱼类。草鱼养殖产业已成为我国淡水鱼养殖产业的重要支柱。然而,作为代表性鲤科鱼类,草鱼同样具有数量显著、形态复杂的肌间刺,这严重限制了草鱼的消费选择和深加工,也制约了其种业与养殖业的高质量发展。此前,本课题组鉴定到了鱼类肌间刺形成的关键调控基因runx2b,并利用基因敲除技术分别构建了斑马鱼（Danio rerio）与团头鲂（Megalobrama amblycephala）的无肌间刺突变品系。但截至目前,尚不清楚runx2b基因是否对草鱼肌间刺的发生发育也起着保守性的调控作用。据此,本研究以草鱼为研究对象,利用传统解剖学方法开展了草鱼肌间刺形态学观察,同时结合不同发育时期草鱼茜素红骨骼染色,探究了草鱼肌间刺形态学特征及其骨化规律。随后,利用CRISPR-Cas9基因编辑技术开展了草鱼runx2b基因编辑实验,成功构建F0代runx2b突变体草鱼。待F0代runx2b突变体草鱼性成熟后,选择runx2b基因均发生有效突变的雌雄个体进行配组繁殖,获得F1代runx2b突变体草鱼。之后,利用茜素红骨骼染色、X-ray扫描、Micro-CT三维成像、组织切片染色等技术方法观察F1代突变体草鱼骨骼表型,筛选获得完全无肌间刺的草鱼新种质。同时通过Sanger测序进行F1代无肌间刺草鱼的基因分型,明确了草鱼肌间刺缺失的遗传基础。主要研究结果如下: 1.草鱼肌间刺形态学特征及其骨化规律研究运用传统解剖学方法对175尾野生型草鱼进行肌间刺形态观察与数目统计分析,结果显示,与其他鲤科鱼类一样,草鱼仅存在髓弓小骨和脉弓小骨2种类型的肌间刺。草鱼的肌间刺数目多且形态复杂,根据其复杂程度可大致分为:“I”形、“(”形、“卜”形、“y”形、“一端多叉”形和“两端多叉”形6种形态。草鱼肌间刺总数介于108-127根之间,平均约118根。研究还发现,草鱼腹腔肋骨处无肌间刺的存在,背侧肌间刺相比于腹侧肌间刺数目多且形态复杂程度更高,身体左右两侧的肌间刺数目接近,无显著性差异（p>0.05）。草鱼尾部肌间刺形态主要为“I”形,随后向头部方向过渡为“(”形或“卜”形,再形成更复杂的其它形态。不同发育时期野生型草鱼肌间刺的骨化特征研究表明:15 dph时,草鱼主轴骨骼与附肢骨骼基本发育完整,但其肌间刺并未出现;约20 dph时,5-10根肌间刺逐渐出现在草鱼尾部区域,形态呈“I”形;约25 dph时,肌间刺开始出现在背鳍下方,伴随着一些过渡形态的“卜”形肌间刺在躯干部出现;约30 dph时,肌间刺生长至背鳍前方,形态复杂的“y”形肌间刺逐渐出现;40 dph后,肌间刺基本全部出现且发育完整。草鱼肌间刺是由尾部向头部逐渐分化的。 2.草鱼runx2b基因突变F0代选育群体构建基于草鱼最新runx2b基因序列在exon4与exon7进行基因编辑靶位点的设计与g RNAs的合成,并利用CRISPR-Cas9基因编辑技术于2021年对I细胞期草鱼受精卵进行胚胎注射,构建了草鱼runx2b基因突变F0代选育群体。胚胎早期靶位点突变有效性检测结果显示,runx2b-exon4-target的突变率为13.3%,runx2b-exon7-target的突变率为26.7%。待F0代突变体草鱼生长至3月龄时,剪其尾鳍提取基因组DNA并利用靶位点特异性引物筛选突变个体,共获得17尾runx2b成功发生突变的F0代草鱼个体,其中在runx2b-exon4-target突变的为4尾个体,而runx2b-exon7-target则17尾个体均有突变,成功筛选出的草鱼runx2b突变个体作为F0代亲本用于繁殖F1代。 3.F1代无肌间刺草鱼个体筛选及其表型分析 F0代亲本经2年强化培育后,性成熟的8尾雄鱼与1尾雌鱼被作为亲本,繁殖获得F1代个体。为分析F1代草鱼个体的肌间刺表型,随机挑选了26 dph的94尾F1代草鱼个体进行茜素红整体骨骼染色,结果显示,5尾F1代草鱼个体（占比5.32%）的肌间刺完全缺失,主轴骨与附肢骨等其他骨骼发育正常。为获得无肌间刺的F1代草鱼群体,当养殖至3月龄时,对所有F1代草鱼个体进行X-ray扫描筛选。结果从8,477尾F1代草鱼个体中筛选出了完全无肌间刺的草鱼1,678尾,占比约19.79%。分别采集无肌间刺个体以及野生型草鱼,固定后利用Micro-CT进行3D成像分析,结果显示,除肌间刺完全缺失外,无肌间刺个体的其他骨组织密度以及脂肪、肌肉体积均与野生型草鱼无显著性差异（p>0.05）。组织学比较中,分别利用HE染色石蜡切片和Masson染

关键词：草鱼肌间刺 runx2b 基因编辑表型分析基因分型

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