核仁因子Bms1l在斑马鱼肝脏发育过程中的功能研究

发布时间：2018-09-18 07:15

【摘要】：肝脏的发生和发育是一个受到精密调控的复杂过程。人们利用模式生物的研究已经逐渐了解了肝脏细胞的起源、形态发生及生长发育过程,也鉴定出了一些参与肝脏发育的重要因子。作为一种研究胚胎早期发育理想的模式生物,斑马鱼受到了越来越多研究人员的关注,尤其是结合目前迅速发展的CRISPR-Cas9等技术,使得相关的研究手段更加多样、层次更加深入。本课题综合利用遗传学、发育生物学、分子生物学和细胞生物学的研究方法,以斑马鱼为模式生物,系统研究了bms1l基因在斑马鱼早期胚胎发育过程中的作用。通过ENU化学诱变筛选小肝脏表型的斑马鱼,我们课题组获得了一个bmsll基因突变的斑马鱼品系bmsll~(sq163)。研究显示在bmsll~(sq163)突变体中,bmsll基因第5外显子发生了T到A的碱基突变,从而导致bmsll第154位密码子从亮氨酸(CTG)置换为谷氨酰胺密码(CAG)。相关表型分析发现bmsllsql63突变体中,包括肝脏、外分泌胰腺和肠道在内的消化器官生长发育受到特异性的阻滞。进一步研究显示,肝脏在早期起始和分化阶段并没有受到影响。为了深入验证bmsll在肝脏发育过程中的重要作用,我们利用CRISPR-Cas9技术对bmsll基因进行敲除,从而获得了另一个品系的突变体bms1lzjul。在该突变体中,bmsll基因第2个外显子上有13个碱基的插入,从而破坏了蛋白的正常编码。由于Bmsll蛋白的完全缺失,bmsllzju1突变体有着更为严重的消化器官生长发育缺陷。我们在bmsll~(sq163)和bmsllzju1两个等位基因突变体的基础上深入研究了Bmsll在斑马鱼肝脏发育过程中的功能。前人在酵母的研究中发现Bmsl是一个核仁蛋白并且能与Rcl1蛋白形成复合物,共同参与核糖体前体RNA(pre-rRNA)的加工剪切。因此,Bmsl对18S rRNA的成熟及核糖体生成有重要作用。根据以上研究,我们想了解斑马鱼中的同源基因bmsll是否有这些保守的功能。首先,通过免疫荧光共定位及免疫共沉淀实验,我们发现Bmsll和Rcl1都是核仁蛋白并且能够互作,同时也用人的这两个蛋白进一步验证了该功能的保守性。其次,我们发现bmsll~(sq163)和bmsllzjul突变体中pre-rRNA的加工过程和18S rRNA的生成均受到了影响,说明Bmsll确实在其中行使重要功能。另外,突变体肝脏细胞的核仁由于受到异常的pre-rRNA转录和加工影响,其形态也发生了明显的改变。在上述研究过程中,我们发现bmsll突变体核仁中rDNA的转录水平显著上升,这使得与转录方向相反的DNA复制叉受到严重阻滞,进而引起了DNA损伤反应。我们详细研究了其中的P53信号通路,结果显示在bmsll突变体中,P53及其异构体的表达水平显著提高,并且受P53调控的下游基因表达也被激活。由于P53的活化能够抑制细胞周期的进行,我们在P53突变的背景下分析了bmsll~(sq163)突变体中消化器官的拯救情况。结果显示该双重突变体能够部分拯救小肝脏和小胰腺的表型。接着,我们详细分析了bmsll~(sq163)突变体的肝脏细胞在细胞周期中具体受阻滞的时期。结合细胞周期标志蛋白免疫荧光和Edu掺入实验,我们发现突变体中处于S期DNA复制阶段的肝脏细胞比例显著上升,相应地,G2期至M期的比例显著下调。通过Edu和Brdu不同时间点的双标实验,我们进一步验证了bmsll突变体的肝脏细胞中存在旺盛的DNA复制现象,从而使得肝脏中非整倍体和多倍体细胞的比例上升。综合上述在bmsll~(sq163)和bmsllzju1突变体中的研究,我们不仅验证了Bmsll是一个功能保守的蛋白,也发现了Bmsll在斑马鱼肝脏发育过程中有重要作用。此外,关于细胞周期和rDNA转录、复制的研究也让我们更系统、全面地了解突变体中发生的关键事件,为我们下一阶段更深入探索Bmsll的功能和作用机制打下了坚实的基础。
[Abstract]:The genesis and development of the liver is a complex process regulated precisely. The origin, morphogenesis and development of liver cells have been gradually understood by using model organisms. Some important factors involved in liver development have also been identified. With the rapid development of CRISPR-Cas9 and other technologies, more and more researchers have paid close attention to it, which makes the related research methods more diversified and deeper. The role of ms1l gene in the early embryonic development of zebrafish was studied. A zebrafish strain bmsll ~ (sq163) with bmsll ~ (sq163) gene mutation was obtained by ENU chemical mutation screening of zebrafish with small hepatic phenotype. The results showed that mutation of the base T to A occurred in exon 5 of bmsll ~ (sq163) mutant, which led to the mutation of bmsll ~ (sq163) gene. The 154th codon of bmsll was replaced by glutamine code (CAG) from leucine (CTG). Phenotypic analysis revealed that the growth and development of digestive organs, including liver, exocrine pancreas and intestine, were specifically blocked in bmsllsql63 mutants. Further studies showed that the liver was not affected in the early initiation and differentiation stages. In order to further verify the important role of bmsll in liver development, we used CRISPR-Cas9 to knock out the bmsll gene and obtained another mutant bms1lzjul. In this mutant, there were 13 bases inserted into the second exon of the bmsll gene, which destroyed the normal coding of the protein. Bmsllzju1 mutant was found to be a nucleolar protein and to be capable of producing Rcl1 eggs. Bmsl plays an important role in the maturation and ribosome formation of 18S rRNA. Based on the above studies, we want to know whether the homologous gene bmsll in zebrafish has these conservative functions. First, through immunofluorescence co-localization and immunoprecipitation experiments, I We found that Bmsll and Rcl1 were both nucleolar proteins and could interact with each other, and the conservativeness of the function was further verified by human proteins. Secondly, we found that the processing of pre-rRNA and the production of 18S rRNA in bmsll ~ (sq163) and bmsllzjul mutants were affected, suggesting that Bmsll did play an important role. In addition, the morphology of the nucleolus of the mutant hepatocytes was significantly altered by abnormal pre-rRNA transcription and processing. During the above studies, we found that the transcriptional level of rDNA in the nucleolus of the bmsll mutant increased significantly, which resulted in severe blockade of the DNA replication fork opposite to the transcriptional direction and consequently caused DNA damage. We studied the P53 signaling pathway in detail. The results showed that the expression of P53 and its isomers was significantly increased in bmsll mutants, and the downstream gene expression regulated by P53 was also activated. The results showed that the double mutant could partially rescue the phenotype of small liver and small pancreas. Then, we analyzed the specific phase of cell cycle arrest in bmsll ~ (sq163) mutant hepatocytes. The proportion of hepatocytes in S-phase DNA replication phase increased significantly, and the proportion of G2 to M-phase decreased significantly. By double-labeling experiments at different time points in Edu and Brdu, we further verified that there was exuberant DNA replication in the hepatocytes of bmsll mutants, which resulted in aneuploidy and polyploid cells in the liver. Based on the above studies in bmsll ~ (sq163) and bmsllzju1 mutants, we not only verified that Bmsll is a functional conserved protein, but also found that Bmsll plays an important role in zebrafish liver development. In addition, the study on cell cycle and rDNA transcription, replication also allows us to understand the mutation more systematically and comprehensively. The key events in Bmsll lay a solid foundation for us to explore the function and mechanism of Bmsll in the next stage.
【学位授予单位】：浙江大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：Q954.4

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