| Hematopoietic and endothelial lineages play a significant role in the course of embryonic development. A lot of diseases that human beings suffer from are closely related to the abnormal development of hematopoietic or endothelial lineages, both of which originate from a same cell, called as hemangioblast. Hemangioblast comes from mesoblast and could differentiate to hematopoietic progenitors and vascular progenitors. The primitive hematopoietic cells produced by hematopoietic progenitors take part in primitive hematopoiesis, while hematopoietic stem cells formed by hematopoietic progenitors regulate and control the definitive hematopoiesis to generate mature erythrocytes, myeloid cells and lymphocytes. Vascular progenitors mainly differentiate to vascular endothelial cells and at the end form arteries, veins, lymphatic vessels etc. The whole development process of hematopoietic and endothelial lineages proceed under the adjustment and control of a complicated but precise molecular signal network. And any abnormity of the key molecular adjustment and control could lead to severe developmental defects or serious diseases. Till now few knowledge of the molecular mechanism that adjusts and controls the development of both hematopoiesis system and hematopoietic system has been disclosed. Therefore, it helps build up the molecular signal network in the course of hematopoietic and blood vessel development to study the key molecular in the process of hematopoietic and blood vessel development, thus providing new molecular targets and treatment plans to some related diseases.The research studies the effect of a new gene named HFHG45whose functions has not been reported on the formation and differentiation of hemangioblasts. In-situ hybridization and RT-PCR were respectively conducted in the zebrafish’s embryos of each development stage to probe the spatial-temporal expression profile of gene HFHG45. The analysis results showed that gene HFHG45was widely expressed in zebrafish, especially in its endothelial lineages. Strain TG (HFHG45:egfp) of genetically modified green fluorescence zebrafish with HFHG45promoter was established to more intuitively observe the expression of gene HFHG45. The analysis results revealed that the zone where green fluorescence presented was near the zone where the in-situ hybridization probe expressed. HFHG45polyclonal antibody of zebrafish was prepared to study the expression of HFHG45protein. According to the Western-blot result, HFHG45protein was expressed in each development stage of the zebrafish’s embryo. In addition, it was expressed in the heart, hematopoietic, brain and muscular tissue of mature zebrafish. It was especially obviously expressed in the hematopoietic. All those results hint that gene HFHG45might be related to the development of zebrafish’s hematopoietic. Making use of the specificity of gene HFHG45of Morpholino in zebrafish to knock down the expression of the gene, it was showed that there were abnormal development of zebrafish in terms of endothelial lineages and hematopoietic. TALEN knock-out technology was employed to get3knock-out strains of zebrafish with gene HFHG45to lucubrate the biological functions of gene HFHG45. The analysis of the death of knock-out strain F2proves that death by mutation might be caused by the knock-out of homozygote on gene HFHG45. An analysis of the phenotype of the knock-out strains exposed that the development of intersegmental endothelial lineages and caudal arteries and veins of the zebrafish was restrained with the amount of hemocytes dramatically decreasing. The result indicates that gene HFHG45may play a quite important role in the formation of zebrafish’s hematopoietic and endothelial lineages. How does the gene affect the development of zebrafish’s hematopoietic and endothelial lineages? Initially, whether or not HFHG45affects the primitive hematopoiesis of zebrafish should be researched. The marker gene scl, mop, lcpl and hbbe3in the tested hematopoietic of in-situ hybridization of embryo, the marker gene gatalin primitive erythrocyte progenitors and the marker gene pu.1in primitive myeloid cells progenitors were remarkably less expressed after the knock-out of homozygote in HFHG45. And O-dianisidine dye showed that hemoglobin was also less expressed after the knock-out of homozygote in HFHG45. All these results manifest that HFHG45may directly adjust and control the development of primitive hemocytes. Then the effect of HFHG45on zebrafish’s definitive hematopoiesis was studied. The in-situ hybridization result of marker gene ragl of lymphocytes demonstrated that ragl was barely expressed in the knock-out homozygote of HFHG45of zebrafish with the amount of mature myeloid cells decreasing. It was discovered through the dye of neutral red and Sudan black that the amount of macrophages and nuetrophile granulocytes formed by the differentiation of myeloid cells was influenced. An analysis is about the in-situ hybridization of such marker genes as cmyb, etc. In hematopoetic stem cells and the knock-out of HFHG45in TG(cmyb:egfp, kdrI:mCherry) double genetically modified zebrafish revealed that there were no green fluorescence of marker cmyb hematopoetic stem cells or red fluorescence of marker kdrl endothelial cells in the zebrafish’s dorsal aorta. The experiment results prove that the knocked-out homozygote in HFHG45originate from the endothelial cells that could not properly form hematopoietic stem cells in the zebrafish’s dorsal aorta. Thus, it is certified that HFHG45might be necessary for zebrafish’s definitive hematopoiesis. To study the effect of HFHG45on the endothelial cells of zebrafish’s endothelial lineages, the two trains TG (flila:egfp)and TG (kdrl:mCherry) of genetically modified zebrafish were used to analyze the development of their endothelial lineages under the circumstance that gene HFHG45was knocked out. It is intuitively and clearly revealed through the fluorescence expression that the knock-out of HFHG45resulted in the formations of zebrafish’s intersegmental endothelial lineages, caudal aorta and caudal veins being distinctly restrained. The in-situ hybridization results of the marker gene flkl, tie2and flila in endothelial cells suggest that the knock-out of homozygote in gene HFHG45lead to a considerable decline in them. All the results confirm that gene HFHG45may play an indispensable role in the formation of zebrafish’s endothelial lineages.It is known that zebrafish’s hemocytes and vessel endothelial cells all originate from hemangioblasts. Then does gene HFHG45affect the development of hematopoietic and endothelial lineages through directly affecting the development of hemangioblasts? The in-situ hybridization of marker genes scl, gata2, etv2and gata5in hemangioblast indicated that the development and differentiation of the embryo and hemangioblasts of zebrafish with its gene HFHG45having been knocked out were severely influenced. The experiment results suggest that gene HFHG45might affect the formation of hemangioblasts, then by which to generate influence on the formation of vessels.Besides, looping abnormity was also observed at72h in the heart of the zebrafish with gene HFHG45being knocked out. A physiological analysis on its heart revealed that its heart’s function was affected. Then does gene HFHG45directly take part in adjusting and controlling the development of zebrafish’s heart, or is it an indirect influence caused by the abnormal development of hematopoietic and endothelial lineages? The RT-PCR result of the earlier marker gene showed that knock-out of gene HFHG45made no obvious difference in the expression of marker genes nkx2.5, tbx5of heart progenitor cells. The in-situ hybridization results of marker gene amhc in atrium, marker gene vmhc in ventricle and marker gene myl7and bmp4in heart also indicated no change in the expression of these genes. It is speculated that gene HFHG45might indirectly adjust and control the development of zebrafish’s heart on the basis of those results.The research results suggest that gene HFHG45in zebrafish might be a necessary regulator of controlling the formation and differentiation of hemangioblasts. It may take part in adjusting and controlling the development of zebrafish’s hemopoietic system and hematopoietic vessel system, and be the source gene of the regulating network to adjust and control the formation and differentiation of hemangioblasts. Expounding the molecular regulation mechanism of the gene would contribute to improve the molecular signal network in the development course of vertebrates’hematopoietic and endothelial lineages, and make it possible to provide new treatment target sites in the future clinical treatment of the diseases caused by the development of hemangioblasts.The study on the other two genes-TMP3and Spata34-is also carried out. Gene TMP3may be a gene closely related to the development of heart. Knocking down the expression of TMP3could lead to obvious abnormity in heart. Carrier Tol2was modified to study the effect of the expression of TMP3on the heart:a polyclone sequence with IRES locus was inserted between promoter cmlc2and coding region EGFP to establish the genetically modified expression carrier pTol2-cmlc2-TMP3-IRES-EGFP. Such a kind strain of genetically modified zebrafish with the expression carrier was gained by the zebrafish’s embryo injection. At the same time, gene TMP3was inserted into the carrier to form recombinant plasmid that was injected into the embryo of zebrafish to get the genetically modified zebrafish strain with stable inheritance in the protein expressing green fluorescent. In addition, the polyclonal antibodies of TMP3and its relevant genes TMP4and GEFT were prepared. Here we also cloned a novel gene, Spata34, encoding a LRR containing protein of415aa. Spata34gene consisted of9exons and8introns and mapped to chromosome3qA3. Spata34is conserved across species in evolution. The Spata34gene was expressed at various levels, faintly before first weeks postpartum and strongly from2weeks postpartum in adult testes. Western blot analysis showed that Spata34protein was specially expressed in mouse testis. Immunohistochemical analysis revealed that Spata34protein was most abundant in the cytoplasm of round spermatids and elongating spermatids within seminiferous tubules of the adult testis. Overexpression of Spata34in COS-7cells inhibited the transcriptional activity of AP-1, p53and p21which suggested that Spata34protein may act as a transcriptional repressor in p53and p21pathway. |
PDF Full Text Request