Temporal And Spatial Expression Pattern Of Cd99l2 Gene And Its Relationship With Hematopoiesis During Zebrafish Development

BackgroundHuman CD99 is a ubiquitous 32 kD a transmembrane glycoprotein encoded by a pseudoautosomal MIC2 gene, located in Xp22.33-pter and Yp11-pter,and its function is not fully understood. The preceding reports show that human CD99 is expressed in the cells of bone marrow, thymus and peripheral blood, the level of expression is related to distinct maturational stages, and have a high level in the immature cells. Is the phenomenon of CD99 expressing highly in the immature cells suggests that it plays an important regulatory role in hematopoiesis? We have great interest in this phenomenon.Hematopoiesis is a process of differentiation, development and maturation of various blood cells. The process is a multi-stage, multi-step process and is affected by many factors. There are evidences show that the adult human and mouse hematopoietic stem cells (hematopoietic stem cells, HSCs) is to become a source of hematopoietic cells, a single HSC can be reconstructed to the entire hematopoietic system in a suitable condition. Proliferation and differentiation of human hematopoietic stem cells is conducted in strict procedures, and subjected to many growth regulators. First of all, after hematopoietic stem cells proceeding to self-proliferation, some cells are directed to the common lymphoid progenitor, followed by differentiation into mature peripheral lymphocytes. Another type of cells differentiated into commonmyeloid progenitors, and finally differentiated into mature blood cells such as granulocytes, monocytes, red blood cells and platelets. Although a large number of studies have been committed to the origin, differentiation development, proliferation, maturation of human hematopoiesis, but these is no direct morphology evidence for human hematopoietic stem cell, and regulatory mechanisms is is not fully understood. Mouse is recognized as one of the best model organisms, however, it is the embryonic development of the uterus, difficult for early hematopoiesis development observation. These shortcomings limited to some extent the application for the studies of early embryonic hematopoiesis. In recent years, a model called zebrafish (zebrafish or Danio rerio) quickly attracted the attention of a large number of laboratories and scientists around the world. It owns high homology with the human genome and similar component of blood cells such as red blood cells, granulocytes, monocytes, lymphocytes and platelets. Its hematopoiesis is conserved highly. The zebrafish (Danio rerio) has emerged as an ideal organism for the study of hematopoiesis, the process by which all the cellular elements of the blood are formed. These elements, including erythrocytes, granulocytes, monocytes, lymphocytes, and thrombocytes, are formed through complex genetic signaling pathways that are highly conserved throughout phylogeny. Large-scale forward genetic screens have identified numerous blood mutants in zebrafish, helping to elucidate specific signaling pathways important for hematopoietic stem cells (HSCs) and the various committed blood cell lineages. Here we review both primitive and definitive hematopoiesis in zebrafish, discuss various genetic methods available in the zebrafish model for studying hematopoiesis, and describe some of the zebrafish blood mutants identified to date, many of which have known human disease counterparts. We used zebrafish as a model organism to explore the role of cd99l2(Danio rerio CD99 antigen-like 2, NM₁94369) in the early hematopoiesis, in order to provide more information about CD99 in the process of human hematopoiesis.Furthermore, amino acid sequence of zebrafish cd99l2 has 32% overall identity (42% positive)with human CD99 gene and its transmembrane region shows 58% identity and 98% similarity, which makes it impractical to study the function of zebrafish cd99l2 for the revealing of the human CD99 in blood cell differentiation, development and function. To study cd99l2 function in hematopoiesis during zebrafish development, we should first explore the temporal and spatial expressing pattern of the cd99l2 gene during zebrafish development. As the lack of commercialization cd99l2 antibody, we can not detect the expression of cd99l2 protein. We used whole mount in situ hybridization (WISH) to detect of the expression of cd99l2 at the transcription level. Unlike the in situ hybridization in the slide, the WISH is to detect the whole zebrafish embryos, exporing three-dimensional information of gene expression during the embryonic development. Compared with the hybridization in the sections, the most important feature of WISH is the integrity. It can be more intuitive and comprehensive in the detection of expression site of the gene. Because WISH detects the whole embryo, which contains all of the transcriptions of corresponding stage, requiring higher in the length of probe. To ensure specificity of hybridization, the probe for cd99l2 of zebrafish labeled with digoxingenin-UTP was synthesized in vitro. After ensured the validity of the probe, the temporal and spatial expression cd99l2 was observed in the way of WISH. We discovered a new signal region, which we speculate it may be the site of early hematopoiesis. We introduced three mutants of cloche, scl and moonshine related with hematopoiesis to explore the relationship between cd99l2 and early hematopoietic. cloche, an early acting zebrafish gene, is required by both the endothelial and hematopoietic lineages. It is involved in the genesis and early diversification of the endothelial and blood lineages, possibly by affecting a common progenitor cell population. Mutant of scl resulted in a loss of primitive and definitive hematopoietic cell lineages. Angioblasts were specified normally in the absence of scl, but later defects in angiogenesis were evident. Mutations in the zebrafish moonshine (mon) gene specifically disrupt both embryonic and adult hematopoiesis, resulting in severe red blood cell aplasia. We verified the hematopoietic lineage situation in all three mutants, including erythrocytes (βel, O-dianisidine staining),myeloid (lyc, sudan black staining), lymphocytes (rag 1), vessels and angioblast (flil). Here, we subject the three mutants at different stages of hematopoiesis disorder to explore the relationship between cd99l2 and early hematopoiesis. In this study, we take the advantages of zebrafish to study the role of CD99 in the development especially hematopoiesis through the zebrafish ortholog gene cd99l2 (GenBank accession no. AI942796).Experiments will be conducted in three parts:Chapter one, the preparation of probe for cd99l2 of zebrafish labeled with digoxingenin-UTP1.Objective:In order to study the expression pattern of cd99l2 during zebrafish development, the RNA probes for whole-mount in situ hybridization were prepared in this study.2.Methods:The cd99l2 fragment obtained by RT-PCR was cloned into pGM-T Easy, then plasmids were linearized with the restriction enzymes of Sacâ…¡, using Sp6 RNA polymerase, the digoxingenin labeled antisense probes were synthetized in vitro, and confirmed by whole-mount in situ hybridization.3.Results:The plasmid of cd99l2/pGM-T has been constructed. Antisense probe was synthetized in vitro. cd99l2 gene expression pattern during embryogenesis of zebrafish was examined using antisense probe.4.Conclusions:The antisense probe can be used for spatial and temporal distribution of cd99l2 during zebrafish development.Chapter two Temporal and spatial expression pattern of the cd99l2 genes1.Objective:To define the function of cd99l2 gene in the development through detecting the temporal and spatial expression pattern of the cd99l2 genes.2.Methods:The whole-mount in situ hybridization(WISH) is that it is a quick and efficient method to establish spatial and temporal gene expression patterns in embryos and early larvae.3.Results:we found that cd99l2 strongly express in the central nervous system consistenting with the previously reported. Amazingly, we found a new expression region.4.Conclusions:Compare with the temporal and spatial expression pattern of theβel gene, We conclude that the new region likely to be early hematopoietic region.Chapter three:The role of cd99l2 gene in the hematopoiesis during zebrafish development.1.Objective:To define the function of cd99l2 gene in the development hematopoiesis during zebrafish development2.Methods:Checking the dynamic expression of all lineage markers in hematopoiesis in wild type, cloche, scl and moonshine by WISH, O-dianisidine staining, and Sudan black staining.3.Results:The Phenotypes of cloche, scl and moonshine are consistent with the previous reports, and The expression of cd99l2 were completely lost in cloche, scl, and moonshine mutant, but normally expressed in the wild type.4.Conclusions:cd99l2 is expressed in the erythroid lineage of primitive hematopoiesis but not in other lineage.