脾脏是脊椎动物最重要的免疫器官之一，而海马先天性缺失脾脏，是脊椎动物器官进化史上的特殊事件。Tlx1基因在脊椎动物脾脏发生中起核心调控作用，其功能域高度保守，而三种海马的Tlx1基因发生了共有的突变，导致功能域三维结构变化，影响了原有功能。据此，申请人提出Tlx1基因功能域突变导致海马先天性无脾的假设。本项目拟通过分析Tlx1基因序列特征和蛋白三维结构，研究该突变对其结构和功能的潜在影响；通过原位杂交和免疫组化分析Tlx1基因在海马胚胎期的时空表达模式和作用靶点，研究Tlx1基因在海马胚胎发育中的潜在功能；利用CRISPR/Cas9技术，在模式生物斑马鱼中敲除并定点突变（g. 622A>G）Tlx1基因，研究Tlx1基因在海马胚胎发生中的作用，揭示海马先天性无脾与Tlx1基因突变的相关性。本申请不仅能揭示海马先天性无脾的原因，同时对研究脊椎动物脾脏发生的进化及其基因调控机制具有重要意义。

The spleen is one of the most important immune organs in vertebrates. Seahorse (genus Hippcompus) is congenital absence of the spleen, which is a special event in the evolutionary history of vertebrate organ. Tlx1 gene has been reported to be a key regular in the development of the spleen in vertebrates, and the gene functional domain is much conserved. Based on the results of bioinformatic analysis and biological experiments, the applicants found that the functional domain of Tlx1 gene in three kinds of seahorse occurred a common mutation (p. Ala200Thr), resulting in the change of the structure of the hydrophobic contact area, which affected the function of the gene. Consequently, the applicants proposed the hypothesis that the mutation of Tlx1 gene resulted in seahorse congenital asplenia. This project intends to explore the structural features of Tlx1 gene and protein by bioinformatics analysis and homologous modeling, to study the impact of the mutation on its function. Through in situ hybridization and immunohistochemistry, we will study the spatiotemporal expression pattern and the target of the Tlx1 gene in the embryo, to analyze the potential function of the gene in the development of embryo of seahorse. We will use CRISPR/Cas9 knockout technology to verify the function of Tlx1 gene in spleen occurrence of seahorse, and further use site-directed mutagenesis (g. 622A>G) to clarify the correlation of seahorse congenital asplenia and Tlx1 gene mutation. This application could reveal the reason of seahorse congenital asplenia and make up the blank of the research on the development of fish spleen, and consequently has important implications to study of the evolution of vertebrate spleen and its gene regulation mechanism.