Sox19b基因对斑马鱼胚胎早期神经系统发育的调节及作用机制的研究
发布时间：2018-08-29 17:31
【摘要】：研究目的 SOX (Sry related high mobility group box)基因家族是一类由SRY相关基因构成的基因家族,最初是由Gubbay. J等在小鼠Y染色体中发现的起性别决定作用的转录调控因子,其编码的蛋白都包含保守型较高的高速泳动蛋白(high mobility group proteins, HMG)结构域。在个体的早期发育过程中,sox基因广泛参与调控性别决定、软骨形成、神经系统发育、晶状体形成及内胚层发育等过程。 Soxl9b基因是斑马鱼SOXB1家族中重要成员之一,为母源性表达,囊胚期之前为泛表达；合子型表达开始于75%下包时期,主要在预定神经外胚层区域表达：原肠运动后期,soxl9b在神经板区域特异性的表达；24h时期,ox19b的表达主要集中于中枢神经系统及脊索区域；48h时期,主要表达在间脑、端脑、中脑和延脑区域,其表达模式与小鼠中sox2基因的表达基本一致。上述研究结果提示soxl9b基因可能在神经系统发育的过程中发挥重要的调节作用。 本研究主要是通过抑制soxl9b基因的表达,探索soxl9b基因对于神经系统发育过程的调控作用及机制。目前,对于神经系统发育相关疾病的发病机制及治疗方法尚不成熟。因此,研究soxl9b基因在神经系统发育过程的调控机制将为临床治疗神经系统发生相关的疾病提供新的线索和理论依据。 研究方法 本研究主要是利用斑马鱼为模式生物,通过显微注射吗啉寡聚核苷酸(Morpholinos, MO)的方法抑制soxl9b基因的表达,通过整封原位杂交、RT-PCR、组织切片HE染色、BrdU掺入及吖啶橙染色等方法,研究soxl9b基因在斑马鱼神经系统发育过程中的调控作用及机制。 研究结果 1、soxl9b基因在斑马鱼胚胎早期发育过程中时空表达模式的建立 首先,我们通过RT-PCR检测soxl9b基因在斑马鱼胚胎发育不同时期的表达量,然后通过胚胎整封原位杂交检测soxl9b基因在斑马鱼胚胎发育不同时期的表达情况。结果显示,soxl9b基因为母源性表达的基因,囊胚期之前为泛表达；神经外胚层形成之后,soxl9b主要在神经外胚层区域表达；原肠运动后期,主要表达在神经板区域；神经分化结束时,soxl9b基因主要表达在中枢神经系统以及脊索区域。 2、soxl9b基因对斑马鱼胚胎早期神经系统发育的调节作用 抑制soxl9b基因表达之后,斑马鱼胚胎的神经系统发育过程受到影响。神经诱导阶段,原位杂交的检测结果发现,神经外胚层的标记基因sox3的表达区域明显减小,神经板的标记基因otx2和dlx3b基因的表达也明显减少。神经发生阶段,中前脑的标记基因otx2及原神经基因neurogl基因的表达也明显降低。通过检测神经干细胞sox2及神经前体细胞ascl1b基因的表达情况,结果显示其表达也明显减弱。通过BrdU掺入及吖啶橙染色等方法检测胚胎细胞增殖及凋亡的情况,实验结果显示,抑制sox19b基因的表达,会导致中枢神经系统区域细胞凋亡增加,细胞增殖减弱。 3、soxl9b基因对斑马鱼胚胎眼睛发育的调节作用 抑制soxl9b基因的表达会导致斑马鱼胚胎眼睛发育受到抑制,包括小眼或眼睛缺失,视网膜分层及晶状体的结构异常。视网膜的形成包括视网膜祖细胞的增殖,细胞命运决定,神经元及胶质细胞形成等过程。通过原位杂交及RT-PCR进一步研究发现,参与调控眼区发育的眼区转录因子(eye field transcription factors, EFTFs) pax2a、vsx2及rx3的表达区域及表达量都明显减弱或降低。 4、soxl9b基因在斑马鱼胚胎早期神经系统发育过程中作用机制的研究 脊椎动物神经系统发育过程依靠外胚层细胞的胞内信号通路之间的相互协调来实现的,包括BMP、FGF以及Wnt信号通路等,这些信号通路之间的协调作用使得机体神经系统和非神经系统分化得以实现。抑制soxl9b基因的表达后,通过RT-PCR检测出BMP及FGF信号通路中部分配体及下游靶基因的表达都明显降低。 研究结论 1、soxl9b基因调节神经诱导及神经发生的过程。 2、soxl9b基因参与调节神经干细胞及神经前体细胞的增殖和凋亡过程。 3、soxl9b基因调控斑马鱼胚胎眼睛的发育。4、soxl9b基因参与调控BMP及FGF信号通路。
[Abstract]:research objective
SOX (Sry related high mobility group box) gene family is a kind of gene family composed of SRY related genes. Originally, SOX (Sry related high mobility group box) gene family is a sex-determining transcription regulator found in Y chromosome of mice by Gubbay. Structural domains. During the early development of an individual, Sox genes are widely involved in the regulation of sex determination, cartilage formation, nervous system development, lens formation and endodermal development.
Soxl9b gene is one of the important members of the SOXB1 family in zebrafish, which is maternal expression and is ubiquitously expressed before blastocyst stage; zygotic expression begins at 75% subcapsular stage and is mainly expressed in the predetermined neuroectodermal region: soxl9b is locally expressed in the late gastrointestinal motility; ox19b expression is mainly concentrated in the middle during 24h. The expression pattern of soxl9b gene in the mesencephalon, telencephalon, mesencephalon and medulla oblongata was similar to that in mice. These results suggest that soxl9b gene may play an important regulatory role in the development of the nervous system.
The purpose of this study is to explore the regulatory role and mechanism of soxl9b gene in the development of nervous system by inhibiting the expression of soxl9b gene. At present, the pathogenesis and treatment of nervous system development-related diseases are still not mature. Therefore, the study of the regulation mechanism of soxl9b gene in the development of nervous system will be the clinical therapeutic spirit. It provides new clues and theoretical basis for diseases related to systemic diseases.
research method
In this study, zebrafish were used as model organisms to inhibit the expression of soxl9b gene by microinjection of Morpholinos (MO). The regulation of soxl9b gene during the development of zebrafish nervous system was studied by in situ hybridization, RT-PCR, HE staining, BrdU incorporation and acridine orange staining. Function and mechanism.
Research results
1, the establishment of spatio-temporal expression pattern of soxl9b gene in zebrafish embryos during the early stage of development.
Firstly, we detected the expression of soxl9b gene in different stages of zebrafish embryo development by RT-PCR, and then detected the expression of soxl9b gene in different stages of zebrafish embryo development by in situ hybridization. After maturation, soxl9b was mainly expressed in the neuroectodermal region, in the late gastrointestinal motility, mainly in the nerve plate region, and at the end of nerve differentiation, soxl9b gene was mainly expressed in the central nervous system and notochord region.
2, the regulation of soxl9b gene on the nervous system development of zebrafish embryos at the early stage.
After inhibiting the expression of soxl9b gene, the development of zebrafish embryonic nervous system was affected. In the neuroinduction stage, the expression of Sox3 gene in the neural ectoderm was significantly decreased, and the expression of Otx2 and dlx3b gene in the neural plate was also significantly decreased. The expression of labeled gene Otx2 and neurogl was also decreased. The expression of Sox2 and ascl1b in neural stem cells and neural precursor cells was also decreased. BrdU incorporation and acridine orange staining were used to detect the proliferation and apoptosis of embryonic cells. The expression of sox19b gene can increase cell apoptosis and weaken cell proliferation in the central nervous system region.
3, the regulation of soxl9b gene on the development of zebrafish embryos' eyes.
Inhibition of soxl9b gene expression may result in inhibition of eye development in zebrafish embryos, including the absence of microphthalmos or eyes, retinal stratification and lens structural abnormalities. Retinal formation includes the proliferation of retinal progenitor cells, cell fate determination, neuronal and glial formation and other processes. Further studies were carried out by in situ hybridization and RT-PCR. It was found that the expression of pax2a, VSX2 and RX3 in the ocular transcription factors (EFTFs) involved in the regulation of ocular development were significantly decreased or decreased.
4, the role of soxl9b gene in the development of nervous system during early stage of zebrafish embryos.
The development of the vertebrate nervous system depends on the coordination of intracellular signaling pathways in ectodermal cells, including BMP, FGF and Wnt signaling pathways. The coordination of these signaling pathways enables the differentiation of the nervous system and the non-nervous system. After inhibiting the expression of soxl9b gene, the expression of soxl9b is detected by RT-PCR The expression of some ligands and downstream target genes in BMP and FGF signaling pathway decreased significantly.
research conclusion
1, the soxl9b gene regulates neural induction and neurogenesis.
2, soxl9b gene is involved in regulating the proliferation and apoptosis of neural stem cells and neural precursor cells.
3, soxl9b gene regulates.4 development in zebrafish embryo eyes, and soxl9b gene is involved in regulating BMP and FGF signaling pathways.
【学位授予单位】：山东大学
【学位级别】：硕士
【学位授予年份】：2013
【分类号】：R338;Q75
本文编号：2211917
[Abstract]:research objective
SOX (Sry related high mobility group box) gene family is a kind of gene family composed of SRY related genes. Originally, SOX (Sry related high mobility group box) gene family is a sex-determining transcription regulator found in Y chromosome of mice by Gubbay. Structural domains. During the early development of an individual, Sox genes are widely involved in the regulation of sex determination, cartilage formation, nervous system development, lens formation and endodermal development.
Soxl9b gene is one of the important members of the SOXB1 family in zebrafish, which is maternal expression and is ubiquitously expressed before blastocyst stage; zygotic expression begins at 75% subcapsular stage and is mainly expressed in the predetermined neuroectodermal region: soxl9b is locally expressed in the late gastrointestinal motility; ox19b expression is mainly concentrated in the middle during 24h. The expression pattern of soxl9b gene in the mesencephalon, telencephalon, mesencephalon and medulla oblongata was similar to that in mice. These results suggest that soxl9b gene may play an important regulatory role in the development of the nervous system.
The purpose of this study is to explore the regulatory role and mechanism of soxl9b gene in the development of nervous system by inhibiting the expression of soxl9b gene. At present, the pathogenesis and treatment of nervous system development-related diseases are still not mature. Therefore, the study of the regulation mechanism of soxl9b gene in the development of nervous system will be the clinical therapeutic spirit. It provides new clues and theoretical basis for diseases related to systemic diseases.
research method
In this study, zebrafish were used as model organisms to inhibit the expression of soxl9b gene by microinjection of Morpholinos (MO). The regulation of soxl9b gene during the development of zebrafish nervous system was studied by in situ hybridization, RT-PCR, HE staining, BrdU incorporation and acridine orange staining. Function and mechanism.
Research results
1, the establishment of spatio-temporal expression pattern of soxl9b gene in zebrafish embryos during the early stage of development.
Firstly, we detected the expression of soxl9b gene in different stages of zebrafish embryo development by RT-PCR, and then detected the expression of soxl9b gene in different stages of zebrafish embryo development by in situ hybridization. After maturation, soxl9b was mainly expressed in the neuroectodermal region, in the late gastrointestinal motility, mainly in the nerve plate region, and at the end of nerve differentiation, soxl9b gene was mainly expressed in the central nervous system and notochord region.
2, the regulation of soxl9b gene on the nervous system development of zebrafish embryos at the early stage.
After inhibiting the expression of soxl9b gene, the development of zebrafish embryonic nervous system was affected. In the neuroinduction stage, the expression of Sox3 gene in the neural ectoderm was significantly decreased, and the expression of Otx2 and dlx3b gene in the neural plate was also significantly decreased. The expression of labeled gene Otx2 and neurogl was also decreased. The expression of Sox2 and ascl1b in neural stem cells and neural precursor cells was also decreased. BrdU incorporation and acridine orange staining were used to detect the proliferation and apoptosis of embryonic cells. The expression of sox19b gene can increase cell apoptosis and weaken cell proliferation in the central nervous system region.
3, the regulation of soxl9b gene on the development of zebrafish embryos' eyes.
Inhibition of soxl9b gene expression may result in inhibition of eye development in zebrafish embryos, including the absence of microphthalmos or eyes, retinal stratification and lens structural abnormalities. Retinal formation includes the proliferation of retinal progenitor cells, cell fate determination, neuronal and glial formation and other processes. Further studies were carried out by in situ hybridization and RT-PCR. It was found that the expression of pax2a, VSX2 and RX3 in the ocular transcription factors (EFTFs) involved in the regulation of ocular development were significantly decreased or decreased.
4, the role of soxl9b gene in the development of nervous system during early stage of zebrafish embryos.
The development of the vertebrate nervous system depends on the coordination of intracellular signaling pathways in ectodermal cells, including BMP, FGF and Wnt signaling pathways. The coordination of these signaling pathways enables the differentiation of the nervous system and the non-nervous system. After inhibiting the expression of soxl9b gene, the expression of soxl9b is detected by RT-PCR The expression of some ligands and downstream target genes in BMP and FGF signaling pathway decreased significantly.
research conclusion
1, the soxl9b gene regulates neural induction and neurogenesis.
2, soxl9b gene is involved in regulating the proliferation and apoptosis of neural stem cells and neural precursor cells.
3, soxl9b gene regulates.4 development in zebrafish embryo eyes, and soxl9b gene is involved in regulating BMP and FGF signaling pathways.
【学位授予单位】：山东大学
【学位级别】：硕士
【学位授予年份】：2013
【分类号】：R338;Q75
【参考文献】
相关期刊论文 前1条
1 景乃禾;盛能印;谢治慧;;BMP信号通路在中枢神经系统发育过程中的作用[J];细胞生物学杂志;2009年01期
，本文编号：2211917
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