The Expression Pattern Of Hox Genes In The Development Of Zebrafish Hematopoietic

From Drosophila to mammals all Hox genes (homeobox gene) contain a highlyconserved DNA-binding domain. The basic function of Hox genes is to control how andwhere parts of the body develop. During vertebrate evolution, Hox genes have co-optedto achieve a variety of functions. Various downstream targets of Hox proteins identifiedwithin the developing central nervous system (CNS) suggest that Hox genes not onlyplay a role in patterning of the CNS during early development, but also contribute tocell specification and identity. There is increasing evidence that Hox transcriptionfactors play a direct role in normal proliferation and differentiation of hematopoieticcells, as well as tissue regneration and turmorigenesis. All hematopoietic progenitorsexpress Hox genes in a pattern characteristic of the lineage and stage of differentiationof the cell. In murine immatured hematpoietic cells, at least22of the39Hox genes areexpressed. Expression of clustered Hox genes, and enforced overexpression andknockout studies have demonstrated that dysregulation of Hox genes can lead to variousdevelopmental abnormalities and malignancies. Hox genes encode DNA-bindingproteins that are deployed in overlapping domains along various body axes duringembryonic development. This sequential activation of Hox genes in temporal and spatialmode, the Hox code, is critical for the proper positioning of segmented structures alongthose axes, which include the vertebrate, the limbs and also, the digestive andreproductive tracts. It remains unknown how Hox genes are regulated to determine theidentity of hematopoietic stem cells and their derivatives which migrate and expressmost Hox genes.Zebrafish as a model organism is widely used in the study of human development anddisease models. It has a number of advantages such astransparent embryos, easy cultivation, esay operation and powerful regenerativecapacity. What the most important is, zebrafish has a complete blood circulatory system, and its hematopoietic genetic network in the evolutionary is highlyconserved with the human’s. Compared to39Hox genes with humans andother mammals, zebrafish has at least48Hox genes. Therefore, zebrafish is an idealmodel to further investigate Hox code in hematopoietic system.In this study, we first use quantitative RT-PCR or qRT-PCR,to examine theexpressions of41Hox genes in30hpf and60hpf of zebrafish embryos and fouradult organs. These adult organs are supposed to closely link to hematopoiesis. Ourresults show that the number and abundance of the Hox gene expression reduces whenthe embryos grow.There are31hox genes expressing at30hpf, and26hox genes at60hpf. In adult organs, five Hox genes are expressed in, liver,24in heart,22in spleen and23in kidney. The majority of the expressed genes belong to the A&B cluster, andmostly are located the the3’end including paralogs1to9. 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 selectthree representative Hox genes and perform RNA in situ hybridizations. We find thatthe expression of Hoxa2b is located in the head area while, Hoxa5a and Hoxa9a arerespectively expressed in the central and caudally part of the embryo of20hpf. In3dpfjuvenile fish, however, the anterior-posterior (AP) patterns disappear. Hoxa2b, Hoxa5aand Hoxa9a genes become expressed in similar domain，localized in the presumptivethymic primordium. In adult, Hoxa2b、Hoxa5a、Hoxa9a genes are mainly distributed inthe red pulp of the spleen, and in the edge of renal tubules. Given that Hox genes aremainly expressed in the lower differentiated stem cells e or precursors, we propose thatthe neighbor of renal tubules and red pulp of the spleen may be the homing sites wherehematopoietic stem cells proliferate and differentiate into hemacytoblasts ormyeloblasts.To verify our hypothesis mentioned above, we finally create the MLL-AF9transgeniczebrafish. MLL-AF9fusion protein has been shown to aggreasively increase the theexpression of Hoxa cluster genes, particularlyof5’ Hoxa cluster genes. Inthis study wedo find higher expression levels of Hoxa2b，a9a，b5a，b5b in transgenic zebrafish. These results further demonstrate that Hox genes are spatotemporally expressed inhematopoeitic cells and their homing locations.In conclusion, the Hox code refers to the expression pattern of Hox genes in a modeof time and spatial collinearity. This characteristic expressionm play critical roles inin the hematopoietic developemnt of the zebrafish. It is still unknown whether Hoxexpression pattern determines the homing and trifficking og hematopoietic cells orhoming microenviroment confers Hox expression patterns to hematopoietic cells.Whatever, our study has proivided a new venue for us to further validate the functionsof Hox gene in the migration and homing of adult stem cells.