Gfi1aa在斑马鱼造血系统发育中的功能和作用机制研究

The establishment of hematopoietic system is a pivotal physical process in vertebrate development involving a hierarchy regulatory network orchestrated by multiple lineage-specific transcription factors. Mounting evidence suggests mutation or deletion in key transcription factors suffices to result in the abnormity of hematopoietic development. Handful of key transcription factors have been identified and characterized in some depth, but the underlying regulatory genetic networks remain poorly elucidated. In this study, we established Gfi1aa-/- zebrafish model using TALEN genome editing technology. The phenotype analyses revealed knocking out of Gfi1aa in zebrafish blocked the primitive erythrogenesis and erythroid proliferation while the definitive erythrogenes is not affected. Based on this, we hypothesized that Gfi1aa may participate in governing the differentiation and mature course of primitive erythrocytes. In the following study, we will go further to unravel the regulatory mechanism of Gfi1aa through integration of cell biology, genetics and molecular methods to understand the functions of Gfi1aa in primitive erythrogenesis, discover its downstream effectors and figure out the crosstalks between Gfi1aa and Gfi1b, another key transcription factors in hematopoietic development. Fulfill of this project will definitely enlarge our appreciation of the molecular mechanism in erythrogenesis, and it could provide a foundation for researches into exploring underlying mechanisms of erythroid-related diseases.

造血系统发育是脊椎动物发育的重要生理过程，单因子突变或缺失就可能造成造血系统发育紊乱，所以完善造血系统发育的调控网络是亟待解决的科学问题。我们建立了Gfi1aa敲除的斑马鱼模型并分析其表型，发现该突变体原始造血过程中的红细胞发育受到阻遏，红细胞增殖受阻，而晚期的红细胞发生没有受到任何影响。此外我们还发现红细胞分裂相关基因Band3和血红蛋白亚基βe3基因发生了显著性下调，提示Gfi1aa可能通过调控band3和βe3的正常表达从而维持红细胞的正常功能。为了进一步探索Gfi1aa对原始红细胞分化成熟的作用机理和调控机制，本项目将从细胞学，遗传学和分子生物学角度揭示Gfi1aa的作用，弄清其如何调控原始红细胞发育，完善Gfi1aa的下游调控网络，以及明确其在遗传通路中与转录因子Gfi1b的相互作用关系。该项目有助于揭示红细胞发育的分子机制，并为探索红细胞相关血液系统疾病的发生机理提供研究基础。

独立生长因子（Growth factor independence 1, Gfi1）是一种在多种造血细胞中发挥重要作用的调控因子。Gfi1aa是其在斑马鱼中的同源基因。用Morphlino在斑马鱼中敲减Gfi1aa后原始红系造血发生缺陷而髓系造血活跃。利用转座子介导的斑马鱼基因捕获技术造成Gfi1aa缺失后原始造血并没有发生变化。至此，关于Gfi1aa在胚胎造血系统发育过程中的作用还没有得到充分的认识，因此需要一个可稳定遗传的突变体来确定其功能。所以，我们利用TALEN技术建立了Gfi1aa敲除斑马鱼模型。我们的研究发现Gfi1aa在原始造血的红细胞发生过程中不可或缺，但是原始髓系细胞生成在Gfi1aaKO敲除突变体中是正常的。有意思的是，在接下来的髓系分化中，Gfi1aa缺失导致粒细胞增加，而巨噬细胞发育正常。我们的研究证实了Gfi1aa在斑马鱼造血发育过程中的作用。我们还从细胞，分子和遗传关系三个层次上研究了Gfi1aa调控红细胞发育的作用机制。结果发现Gfi1aaKO敲除突变体中红细胞增殖受阻，Band3基因表达显著下降，进一步染色质免疫共沉淀实验证明Band3基因是Gfi1aa的直接下游调控因子。此外我们通过对Gfi1aaKO/Gfi1bKO双敲突变体的表型进行研究，发现该突变体表现出更为严重的红细胞发育缺陷，这提示Gfi1b可能作为Gfi1aa的协同调控因子参与斑马鱼原始造血中的红细胞发育。我们的研究结果完善了斑马鱼原始红细胞发生的调控网络并为了解相关疾病的发生机理提供了理论基础。