Leg1在斑马鱼肝脏发育过程中的功能研究

发布时间：2018-11-20 11:46

【摘要】：与哺乳类动物和鸟类的胚胎发育不同,硬骨鱼类基本上是体外受精体外发育,其肝脏发育过程中会遇到内在或外在压力,因此需要启动一些机制消除或者中和外界压力诱导的有害物质,使得胚胎发育处在最佳的外界环境中。但是当环境压力太大,体内机制不足以保护正常的胚胎发育时,就会导致细胞周期受阻、细胞死亡或者衰老。因此,在长久进化过程中,硬骨鱼类需要一套完善的抗压保护机制来保护胚胎发生和器官形成。而这种抵抗外界环境的机制或者方式是发育生物学有志探究却又知之甚少的领域。legl (liver enriched gene 1,肝脏富集基因1)是一个功能未知的基因。leg1在斑马鱼基因组中存在legla和leglb两个拷贝,序列比对发现leg1a和leglb两个基因,结构相近、序列相似、串联定位在第20号染色体上。两个基因都编码着一条361个氨基酸多肽,两条多肽高度相似,一致性达90.6%。leg1编码一种新型的分泌蛋白,该蛋白包含一个功能未知的结构域DUF781 (domain of unknown function 781),序列分析发现该蛋白在脊椎动物中是保守的。我们采用TALEN技术获得了两个legla突变体品系,即leglzju1和leglzju2。通过整胚原位杂交方法我们观察了两个突变体中肝脏发育情况,发现legla母源-合子型突变体仅在压力条件下(如高密度高温或UV照射或H2O2处理)表现出小肝脏表型。由于在UV照射或H2O2处理条件下同时添加抑制ROS产生的化合物APO和DPI能挽救以上压力条件下legla母源-合子型突变体的小肝脏表型,说明这些压力条件下ROS的过量产生是导致小肝脏表型的原因。用肝脏早期发育的分子标记如foax3, gata6, prox1以及hhex做整胚原位杂交显示,在压力条件下Legla的缺失导致了肝脏细胞周期严重阻滞,从而使得肝芽的形成受到了影响,并最终致使leglazjul母源-合子型突变体的肝脏明显小于野生型的。有趣的是我们发现Legla的缺失对其它消化器官的发育影响不明显,仅外分泌胰腺发育受到些微抑制。在Legla作用机理研究方面,我们通过legla mRNA注射到鱼卵发现在压力条件下Legla促进Erk的磷酸化,通过将表达Legla的质粒转染培养的人细胞发现Legla可能与FGFR3相互作用。此外,我们发现,压力条件下,Legla第70位天冬酰胺(N70)糖苷化修饰突变为丙氨酸后不能激活Erk信号通路,不能保护肝脏发育,同时N70连接的糖苷化修饰对于Legla与FGFR3相互作用非常重要。综上所述,本课题研究发现,Legl作为一个分泌到细胞外的新型信号因子,通过激活Erk信号通路形成了一种新的抵抗压力的信号通路来保护肝脏发育。该研究结果及进一步的深入研究有望揭示硬骨鱼类适应环境改变的原因。
[Abstract]:Unlike the embryonic development of mammals and birds, bony fish are basically developed in vitro during in vitro fertilization, and their liver is subject to internal or external pressure during development. So it is necessary to activate some mechanisms to eliminate or neutralize harmful substances induced by external pressure, so that embryo development is in the best external environment. But when the environment is too stressful to protect normal embryonic development, it can lead to cell cycle arrest, cell death or aging. Therefore, in the long evolution process, bone-fish need a set of perfect pressure protection mechanism to protect embryogenesis and organogenesis. And the mechanism or way to resist the outside environment is. Legl (liver enriched gene 1, a field that developmental biology wants to explore but knows little about. There are two copies of legla and leglb in the genome of zebrafish leg1. Two genes, leg1a and leglb, are found to be similar in structure and sequence by sequence alignment, and they are located on chromosome 20 in tandem. Both genes encode a 361 amino acid polypeptide, and the two peptides are highly similar. The consensus 90.6%.leg1 encodes a novel secretory protein containing an unknown domain, DUF781 (domain of unknown function 781). Sequence analysis showed that the protein was conserved in vertebrates. Two legla mutants, leglzju1 and leglzju2., were obtained by using TALEN technique. The development of liver in two mutants was observed by in situ hybridization. It was found that legla mother-zygote mutants showed small liver phenotype only under high pressure (such as high density high temperature or UV irradiation or H2O2 treatment). The small liver phenotype of legla mother-zygote mutant could be saved under the condition of UV irradiation or H2O2 treatment by adding the compounds APO and DPI to inhibit the production of ROS. It is suggested that excessive production of ROS under these stress conditions is responsible for the phenotype of small liver. In situ hybridization using molecular markers such as foax3, gata6, prox1 and hhex for early liver development showed that the absence of Legla under pressure resulted in a severe arrest of liver cell cycle, which affected the formation of liver buds. Finally, the liver of leglazjul mother-zygote mutant was significantly smaller than that of wild-type mutant. Interestingly, we found that the absence of Legla had little effect on the development of other digestive organs, with only a slight inhibition on the development of exocrine pancreas. In the study of the mechanism of Legla, we found that Legla promoted the phosphorylation of Erk under pressure by injecting legla mRNA into fish eggs, and that Legla might interact with FGFR3 by transfecting the plasmid expressing Legla into cultured human cells. In addition, we found that Legla 70th position asparagine (N70) glycosylation mutated to alanine could not activate Erk signaling pathway and protect liver development under pressure. At the same time, the glycosylation modification of N 70 junctions is very important for the interaction between Legla and FGFR3. In conclusion, we found that Legl, as a novel extracellular signal factor, protects liver development by activating the Erk signaling pathway to form a new stress-resistant signaling pathway. The results and further studies are expected to reveal the reasons for the adaptation of bony fish to the environment.
【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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