位杂交试验。
我们发现在20hpf胚胎Hoxa2b表达在头部，Hoxa5a和Hoxa9a分别在中部和尾部。
但在3dpf幼鱼，这种前后表达模式消失。
Hoxa2b、Hoxa5a和Hoxa9a 都在假想的斑马鱼胸腺原基高表达。
在成体，Hoxa2b、Hoxa5a、Hoxa9a基因主要分布在脾红髓中，白髓中少量分布。
在肾脏，Hoxa2b、Hoxa5a、Hoxa9a基因主要分布在肾小管边缘及肾小管之间的间隙里。
由于Hox基因主要表达在分化程度较低的干细胞或者祖细胞中，因此我们推测Hoxa2b、a5a、a9a在肾脏和脾脏的表达部位可能反映了肾小管附近和脾红髓为斑马鱼肾脏造血干细胞增生并向髓样先祖细胞分化的场所。
为了验证上述假说，我们最后制备了MLL-AF9转基因斑马鱼。
MLL-AF9融合蛋白已被证明能进行增加髓样先祖细胞A簇Hox基因，特别是5`端Hox基因表达。
本研究我们发现Hoxa2b、a9a、b5a、b5b在转基因鱼相对于正常鱼高表达。
这些结果进一步证明Hox基因在造血细胞和它们的巢居场所呈特征性时空表达模式。
总之，Hox基因的时空表达模式，即Hox密码在Hox造血过程中发挥明显作用。
但到底是Hox基因的表达模式指引造血细胞迁移到造血器官，还是造血器官微环境授予造血细胞的Hox基因表达模式有待继续研究。
无论如何，本课题为进一步研究Hox基因在成体干细胞的迁移和巢居中的作用开辟了新方向。
关键词：同源盒基因，成体干细胞，巢居，实时荧光定量，原位杂交，斑马鱼The expression pattern of Hox genes in the development ofzebrafish hematopoietic systemsABSTRACTFrom Drosophila to mammals all Hox genes (homeobox gene) contain a highly conserved DNA-binding domain. The basic function of Hox genes is to control how and where parts of the body develop. During vertebrate evolution, Hox genes have co-opted to achieve a variety of functions. V arious downstream targets of Hox proteins identified within the developing central nervous system (CNS) suggest that Hox genes not only play a role in patterning of the CNS during early development, but also contribute to cell specification and identity. There is increasing evidence that Hox transcription factors play a direct role in normal proliferation and differentiation of hematopoietic cells, as well as tissue regneration and turmorigenesis. All hematopoietic progenitors express Hox genes in a pattern characteristic of the lineage and stage of differentiation of the cell. In murine immatured hematpoietic cells, at least 22 of the 39 Hox genes are expressed. Expression of clustered Hox genes, and enforced overexpression and knockout studies have demonstrated that dysregulation of Hox genes can lead to various developmental abnormalities and malignancies. Hox genes encode DNA-binding proteins that are deployed in overlapping domains along various body axes during embryonic development. This sequential activation of Hox genes in temporal and spatial mode, the Hox code, is critical for the proper positioning of segmented structures along those axes, which include the vertebrate, the limbs and also, the digestive and reproductive tracts. It remains unknown how Hox genes are regulated to determine the identity of hematopoietic stem cells and their derivatives which migrate and express most Hox genes.Zebrafish as a model organism is widely used in the study of human development and disease models. It has a number of advantages such as transparent embryos, easy cultivation, esay operation and powerful regenerative capacity. What the most important is, zebrafish has a complete blood circulatorysystem, and its hematopoietic genetic network in the evolutionary is highly conserved with the human’s. Compared to 39 Hox genes with humans and other mammals, zebrafish has at least 48 Hox genes. Therefore, zebrafish is an ideal model to further investigate Hox code in hematopoietic system.In this study, we first use quantitative RT-PCR or qRT-PCR,to examine the expressions of 41 Hox genes in 30 hpf and60hpf of zebrafish embryos and four adult organs. These adult organs are supposed to closely link to hematopoiesis. Our results show that the number and abundance of the Hox gene expression reduces when the embryos grow.There are 31 hox genes expressing at 30hpf, and 26 hox genes at 60 hpf. In adult organs, five Hox genes are expressed in, liver, 24 in heart, 22 in spleen and 23 in kidney. The majority of the expressed genes belong to the A & B cluster, and mostly are located the the 3 'end including paralogs 1 to 9. While the genes of a10b，a11a，a11b，c4a，c6b，c11a，c11b，c12b，d4a，d9a，d10a，d11a are transiently expressed, the expression of Hoxa2b，a5a，a9a， b2a，b8a and d3a are stably detected in heart, spleen and kidney. Consistently, Hoxa1a，a2b，a3a，a4a，a5a，a9a，a11a，b5a，b6a，b7a，b8a and d3a are highly expressed in the embryos, spleen and kidney, suggesting that these genes may be specific to hematopoiesis.To determine the spatio-temporal expression patterns of Hox genes, we then select three representative Hox genes and perform RNA in situ hybridizations. We find that the expression of Hoxa2b is located in the head area while, Hoxa5a and Hoxa9a are respectively expressed in the central and caudally part of the embryo of 20hpf. In 3dpf juvenile fish, however, the anterior-posterior (AP) patterns disappear. Hoxa2b, Hoxa5a and Hoxa9a genes become expressed in similar domain，localized in the presumptive thymic primordium. In adult, Hoxa2b、Hoxa5a、Hoxa9a genes are mainly distributed in the red pulp of the spleen, and in the edge of renal tubules. Given that Hox genes are mainly expressed in the lower differentiated stem cells e or precursors, we propose that the neighbor of renal tubules and red pulp of the spleen may be the homing sites where hematopoietic stem cells proliferate and differentiate into hemacytoblasts or myeloblasts.To verify our hypothesis mentioned above, we finally create the MLL-AF9 transgenic zebrafish. MLL-AF9 fusion protein has been shown to aggreasively increase the the expression of Hoxa cluster genes, particularly of 5’ Hoxa cluster genes. In this study we do find higher expression levels of Hoxa2b，a9a，b5a，b5b in transgenic zebrafish.These results further demonstrate that Hox genes are spatotemporally expressed in hematopoeitic cells and their homing locations.In conclusion, the Hox code refers to the expression pattern of Hox genes in a mode of time and spatial collinearity. This characteristic expressionm play critical roles in in the hematopoietic developemnt of the zebrafish. It is still unknown whether Hox expression pattern determines the homing and trifficking og hematopoietic cells or homing microenviroment confers Hox expression patterns to hematopoietic cells. Whatever, our study has proivided a new venue for us to further validate the functions of Hox gene in the migration and homing of adult stem cells.KEY WORDS: Homeobox gene, adult stem cells, Homing, Real-time PCR, in situ hybridization,zebrafish.目录第一章综述 (1)1.1 同源盒基因及Hox密码 (1)1.2 Hox基因家族的主要生理功能。
