Study On The Identification Of Hemocyte Specific Marker BmintegrinaPS3and The Regulation Of Bm Ush In Hematopoiesis

Silkworm is the typical model insects, and all the tissues are surrounded by the circulating hemolymph. According to their different morphology, five types of hemocytes have been identified in silkworm, Bombyx mori:prohemocytes, plasmatocytes, granulocytes, spherulocytes, and oenocytoids. The study of hemocytes in circulatory system and HPO in vitro has been well investigated, but precise lineage relationships among different hemocyte types remain unclear.In the present study, we used molecular marker to monitor the proliferation of hemocytes in vivo. Based on the silkworm database, a graunulocyte specific marker BmintegrinαPS3were found and characterized. By the expression of BmintegrinαPS3, the sources and immune function of granulocytes were investigated. Through the homology search, we cloned and functional analysed the hematopoiesis regulator gene Bm Ush in silkworm. The results are as follows:1. The proliferation of larval hemocytes(1) The proliferation of larval hemocytes in circulatory system:During the first four days of the final instar, hemocyte proliferation gradually increased, while BrdU incorporation attained its highest level on L5D5. Subsequently, the BrdU labelling indices were maintained from L5D6to wandering stage. The double immunofluorescence analysis showed that the circulating hemocytes could symmetrically divide into two new daughter cells.All types of hemocytes in circulatory system, excluding spheruloeytes,could be labelled by BrdU. By the stimulus of heat-inactivated E.coli, the percentage of BrdU positive hemocytes significantly increased. The hemocytes could keep proliferating to produce more new cells to meet the requirement for cellular defence.(2) The structures of HPOs during the final instar:HPOs are surrounded by some fat bodies and are located on the wing discs of larvae. By DAPI staining, the developments of HPOs during5th instar larvae were investigated. The data showed that the sizes of HPOs were gradually increased from L5D1to L5D6, while the size on L5D7significantly decreased because of the hemocytes’discharge into hemolymph, not cell apoptosis.(3) The hematopoietic stem cells and hematopoietic progenitors:To locate the hemocyte precursor cells, the label time was extended to carry the BrdU label retention assay.A small numbers of hemocytes retained intense BrdU signals and were termed long-term label-retention cells which were be putative stem cells, while there were many short-term BrdU label hemocytes in circulatory hemolymph which would be hematopoietic progenitors. At the same time, we did BrdU label retention analysis in HPO and found that the hemocytes in HPO actively divided and continually discharge the proliferating hemocytes into hemolymph. So there were short-term BrdU label hemocytes in HPO which would be hematopoietic progenitors.2. The molecular marker and cell immune function of granulocytes(1) Granulocyte-specific genes cloning and sequence analysis:By searching the silkworm genome database, we find BmintegrinαcPS3was highly and specifically expressed in hemocytes. Through the known sequence data and RACE, the BmintegrinαPS3cDNA including the complete ORF sequence was cloned. BmintegrinaPS3locates on chromosome10, the DNA total length is25988bp, containing25exons and24introns. The ORF length is2895bp, encoding965aa, molecular weight is106.5kDa, and isoelectric point is5.16. BmintegrinocPS3contains5conserved integrin alpha domains, has a length of20amino acid signal peptide at the N-terminus, and a typical transmembrane domain at the C terminus. The3D structure of BmintegrinαPS3was predicted to be bent that is very similar to the resolution structure of integrins in mammal.The phylogenetic tree of integrin homologues from different species showed that BmintegrinaPS3was closer to the PS3family.(2) The mRNA expression pattern of BmintegrinαPS3in silkworm: BmintegrinαPS3was expressed at a high level in hemocytes and at a very low level in head. During each period of the fifth instar, from the4th instar molting stage expression level of BmintegrinαPS3began gradually increasing, until L5D6its expression levels reached the highest, and from L5D7to W2, its expression maintained high levels.(3) The subcellular location of BmintegrinocPS3in hemocyte:The BmintegrinocPS3fusion protein was prokaryotic expressed in E.coli, then was affinity purified. Antiserum was produced by immunizing rabbits and was used to detect induced fusion protein. The results proved that antiserum specifically recognized the BmintegrinαPS3fusion protein. Western blotting analysis was carried out in hemocytes with antiserum, and the results showed that BmintegrinocPS3had two different forms:a larger form expressed in cell cytoplasm and a smaller form expressed on cell membrane. Using antiserum for immunofluorescence, we found that the BmintegrinaPS3can specifically recognize granulocytes and mainly distributed on the cell membrane.(4) The activation of BmintegrinaPS3:To further characterize the BmintegrinaPS3protein, the full-length expression vector was constructed and transfected into BmE-SWU3cell line. The results showed that BmintegrinαPS3protein uniformly distributed in cell cytoplasm of BmE-SWU3cells. When three Bmintegrinp full-length expression vectors were respectively cotransfected with BmintegrinocPS3full-length expression vector, BmintegrinαPS3could be gathered to where β subunit existed. Especially, the6150β subunit could gather all the BmintegrinαPS3molecule that one cell expressed. That suggested that the interaction between BmintegrinαPS3and6150β subunit was closer than other two β subunits. BmintegrinocPS3and6150β subunit could form dimmer on cell membrane. All the above, BmintegrinαPS3could exist in two forms:one form expressed in cell cytoplasm when no β subunit and other factors; the other form cutted by some factors and gathered on cell membrane by β subunit.(5) The source of silkworm granulocytes:The RT-PCR and Real-time PCR showed that BmintegrinαPS3expressed from7th day of embryo stage and maintained a high expression level untill the end of embryo stage. It suggests that the granulocytes begin to be produced from precursor cells on7th day of embryo stage and exist in circulatory hemolymph. Through the culture of HPO in vitro and loss of HPO in vivo, it shows that a small part of hemocytes from HPO were differentiated into granulocytes in hemolymph.(6) The immune function of silkworm granulocytes:Injection of bead could induce the encapsulation response by the hemocytes. During the immune response, the expression of BmintegrinαPS3was upregulated at3h, maintained a significantly higher expression levels at12h, and back to the normal level at24h. With the same method, injection of heat-inactivated E.coli could induce the phagocytosis response by the hemocytes. The expression of BmintegrinαPS3upregulated a lot at6h, down regulated weekly at12h, and already close to normal level at24h. All these results indicated that granulocytes as the executor of cell immune response, participate into encapsulation and phagocytosis in different speed.3. Cloning and functional analysis of the hematopoiesis regulation factor BmUsh in silkworm(1) Cloning and sequence analysis of BmUsh:BmUsh cDNA including the complete ORF sequence was cloned. BmUsh locates on chromosome4, containing3exons and2introns. The ORF length is2409bp, encoding803aa, molecular weight is85.89kDa, and isoelectric point is8.3. BmUsh contains8conserved C2H2zinc domains.The phylogenetic tree of FOG homologues from different species showed that FOGs were conserved from invertebrates to vertebrate. There is one FOG in every insect species and during molecular evolution there are two FOGs in every mammal species. BmUsh was closer to the insect Ush family.(2) The mRNA expression pattern of BmUsh in silkworm:The Real-time PCR with cDNA of different stage of embryo showed the expression of BmUsh decreased gradually from lth day to9th day. The Real-time PCR with cDNA of different tissues on L5D3showed that BmUsh expressed in several tissues, but significantly higher in hemocytes than other tissues. The Real-time PCR with cDNA of different stage hemocyte in5th showed that BmUsh were upregulated at the molting stage, the middle day of larve, and the wandering stage.(3) Identification of Nuclear Localization Signal (NLS) in BmUsh: Ush as transcription factors must be targeted to the nucleus. By sequence analysis,10aa at the450-459amino acids site with4arginine series was predicted to be typical NLS motif. This motif with DsRed was together cloned into transient expression vector, and expressed in BmE-SWU3, while each of the four arginine were site mutated respectively and constructed into vector in the same method. The results showed that the predicted NLS motif was able to target all DsRed molecules to nucleus, but the NLS motif with lth and3th arginine mutation could not target any DsRed molecules to nucleus.(4) The interaction factor of BmUsh:In order to find the interaction factor of BmUsh, the full-length expression vector of BmUsh was constructed and transfected into BmE-SWU3. The BmUsh-DsRed were highly expressed and clustered in nucleus. Accoring to the known NLS site, we mutated the456and458arginines of full-length BmUsh and found that the mutant BmUsh could not enter into nucleus. Direct evidence showed the locus indeed played the role of the nuclear localization signal in the full-length protein of BmUsh. Another hematopoiesis regulation factor BmLz was clone into full-length expression vector and cotransfected with BmUsh and mutant BmUsh. The results showed that BmLz was co-located with BmUsh in same site of nucleus, and the BmLz could gather the mutant BmUsh into nucleus or enter into cell cytoplasma to co-locate with mutant BmUsh. BmUsh and BmLz were simultaneously overexpressed by Bac-to-Bac/BmNPV expression system. The immunoprecipitation assay by recombinant bacmid illustrates that BmUsh and BmLz as a protein-protein interaction performs functions in nucleus.(5) The hematopoiesis regulation function of BmUsh:BmUsh was overexpressed in primary culture of circulating hemocytes with the full-length expression vector of BmUsh. BmUsh protein was distributed in nucleus of hemocytes. Comparision between the control plasmid and BmUsh plasmid, the BmUsh could promote the plasmatocyte differentiating into the oenocytoid. Larvae on L5D5were injected with synthetic double-stranded RNA, and the expression of BmUsh was successful knock down. Meanwhile, the expressions of PPO1, PPO2, PPAE, and BAEE in hemocytes were also down. The melanization response of RNAi larvae was not processed. The overexpression and knowndown assay show that BmUsh regulate the differentiation of oenocytoid.(6) BmUsh involve the immune response:Injection L5D3larvae with heat-inactivated E.coli could induce the phagocytosis response of the hemocytes. The results showed that at6h, the expression level of BmUsh increased20-fold; at12h-24h, the expression level of BmUsh already were close to normal level. All these indicate that BmUsh is involved in the melanization response by regulating the differentiation of oenocytoid.