Functional Study Of Drosophila Blood Cells Developmental Candidate Gene CG8419

Previous researches indicated that the transcription factors and signal pathways regulating hematopoietic cells development are conservative in mammals and Drosophila. Although many of the regulatory factors involved in the blood cell formation have been identified, a lot of them need to be identified and the molecular mechanisms remain to be elucidated. Clarifying the molecular mechanism of the hematopoietic lineage has important clinical significance for prevention of blood diseases.The CG8419 gene(a vertebrate homolog of TRIM45)was cloned and identified in our lab. Bioinformatics analysis showed that the protein encoded by this gene has a RING finger, B-box finger, and coiled-coil structure domain, which jointly form a tripartite motif and an IG-FLMN motif structure domain. Previous studies in our lab suggested that the CG8419 gene might be involved in the development of hematopoietic cells.For further study of the functions of this gene in the development of hematopoietic cell in Drosophila, the temporal and spatial expression pattern of CG8419 gene was determined by in situ hybridization and antibody staining. The results showed that CG8419 expressed weakly in head mesoderm at early and mid embryos, and strongly in salivary glands at later stage. The weak expression was observed in the fat body, central nervous system, trachea, proventriculus and other tissues at later embryonic stage.The P-element knockout method was used to obtain the CG8419 null mutant lines. Western blotting analysis with a CG8419 antibody showed that the CG8419 protein expression was significantly decreased in the two knockout lines, indicating that the two lines are null mutant of the gene. RT-PCR results suggested that the mRNA levels of Srp, Lz and Gcm involved in hematopoiesis were significantly down-regulated in the CG8419 P-element mutant and in knockout mutants. Srp is known the earliest gene that regulates the differentiation of hematopoietic stem cell in Drosophila. We prepared a Srp polyclonal antibody. The results by Western blotting with Srp antibody indicated that the Srp protein expression level was down-regulated in the CG8419 P-element mutant and knockout mutants. These results indicated that CG8419 might be located in the upstream of Srp and involved in the development of hematopoietic cell in Drosophila. Further phonotypical analysis showed that compared with wild-type, the amount of mature crystal cells and various blood cells marked by He gene were decreased in the CG8419 P-element mutant and knockout mutants. These results further suggested that the CG8419 gene is involved in the formation of blood cells in Drosophila.In short, the results above indicated that CG8419 is a new gene involved in the formation and differentiation of hematopoietic stem cells in Drosophila, which may be located in upstream of Srp, the earliest gene that is involved in the differentiation and development of hematopoietic stem cells. The results obtained provide a foundation for further clarifying an important function of CG8419 in the formation and differentiation of hematopoietic stem cell in Drosophila.