Studies On The Function Of Jumu In Drosophila Hematopoiesis And Cellular Immunity

Similarly to the process in vertebrates,hematopoiesis in Drosophila occurs in two stages during development:the first population of hemocytes is derived from the embryonic head mesoderm,the second stage of hematopoiesis occurs during the larval stage in the lymph gland.The lymph gland is an important hematopoietic organ of Drosophila in which the maintenance of hematopoietic progenitor cell fate relies on intrinsic factors and extensive interaction with cells within a microenvironment.At the onset of metamorphosis,the lymph gland breaks down,releasing plasmatocytes and crystal cells into the circulating hemolymph.Drosophila lacks adaptive immunity and relies on multiple innate immune responses,such as humoral and cellular immunity,to defend against invading pathogens.The cellular response is involved in plasmatocytes,crystal cells and lamellocytes.In recent years,the mechanisms underlying hematopoiesis and innate immune response have been intensively investigated and show a high degree of similarity between Drosophila and mammals,thus,Drosophila has been studied as good model for investigating diseases associated with hematopoiesis and immunity.Our previous study showed that adult jumu mutant flies show defective hemocyte phagocytosis and a weaker defense capability against pathogen infection.Moreover,Foxn1,the mammalian ortholog of Jumu,has been shown to regulate the growth and differentiation of thymic epithelial cells in mouse,and Foxn1-knockout mice show severe immunodeficiency and a lack of T-cell development.According to these results,to further investigate the role of Jumu in Drosophila hematopoiesis and innate immune can provide a good theoretical basis on understanding the regulatory mechanisms of Foxnl and the other members of FOX families in the mammalian hematopoiesis and related diseases.In this study,jumu mutants and RNAi lines were used as research material,the differentiation of lymph gland hemocyte precursors were detected by using immunohistochemistry,in situ hybridization,Real-time PCR and Chromatin immunoprecipitation(ChIP).Here,we show that Jumu controls hemocyte differentiation of lymph gland through multiple regulatory mechanisms.Jumu maintains the proper differentiation of prohemocytes by cell-autonomously regulating the expression of Col in medullary zone and by non-cell-autonomously preventing the generation of expanded PSC cells.Jumu can also cell-autonomously control the proliferation of PSC cells through positive regulation of dMyc expression in PSC domain,and the results of ChIP and dual luciferase reporter gene assay show that Jumu regulates the transcription of dmyc by binding to its FKH-binding site.Moreover,we also show that a deficiency of jumu throughout the lymph gland can induce the differentiation of lamellocytes via activating Toll signaling.Here,we further investigated the role of jumu in the regulation of larval circulating hemocyte development and phagocytosis by using pathogens injection,immunohistochemistry and Real-time PCR.The loss of jumu leads to the decreased phagocytosis ability of circulating cells and inhibits the extension of filopodia.The results of Real-time PCR and immunstaining show that both the mRNA levels of phagocytosis receptor genes NimC1,Dscam,peste and draper and fascin which is associated with the formation of actin filopodia and the proteion levels of NimC1,Ena and Fascin are down-regualted in jumu deficient circulating hemocytes.Hemocytes in the S and M phase in the lymph glands and circulating hemocytes of jumu double heterozygotes mutants and jumu RNAi lines show a increased number percentage,however,the severe deficiency of jumu causes the defective spindle formation and cytokinesis during mitosis and consequently leads to the generation of enlarged multinucleate hemocytes.The knockdown of jumu in circulating hemocytes causes the reduced mRNA levels of CycA,png and piwi and increased mRNA levels of CycB and CycD.Moreover,the loss of jumu also autonomously leads to the activation of Toll pathway in circulating hemocytes and fat body.Our findings provide new insights into the mechanistic roles of cytoskeleton regulatory proteins in phagocytosis and establish a basis for further analyses of the regulatory mechanism of the mammalian ortholog of Jumu in mammalian innate immunity.