Study On The Mechanism Of The Effect Of HmgD Overexpression On The Pupation And Hemocytes Development In Drosophila

As an important model organism,Drosophila has made contribution to the research on genetics,molecules biology and developmental biology.In addition,about 2/3 genes of human being which cause disease including genes relative to cancer,can be found in Drosophila as similar genes.The high mobility group proteins gene(HmgD),sited on the right arm of the 2 chromosome 57F10,encodes the Drosophila homologue of high mobility group proteins.HMGD,the most abundant HMG protein in DrsophiIa,was isolated firstly by Bassuk in 1982.It is closest to the HMGB subfamily of mammals structurally and functionally,both having the HMG-box motif and thought to be architectural factors in assembling chromatin.HMGD can bend DNA and facilitate forming the chromatin-protein complexes.In addition,HmgD is maternal-effect gene, and the amount of HMGD is highest in ovaries and early embryos,especially before the matemal-zygotic transition(MZT).A wealth of scientic evidence suggested that H1 can be subsitituted by HMGD involved in assembling chromatin orderly,which reduces the compactness of chromatin packing during the very early development of Drsophila. However,the comparison of the DNA-binding capacity and specificity in recognition of DNA clearly showed that HMGD does not share the binding properties with histone HI. Moreover,HMGD is expressed not only in adult females and early embryos but also during later embryonic,development,suggesting that HMGD may exert other functions than that of a specific early embryonic substitute of H1.These results implicate that HMGD may have other unknown functions than only play a general mitosis-related role during early embryogenesis.To analyse the molecular mechanism of overexpression of HmgD on the pupation and hemocytes development in Drsophila,we have generated pUASpHmgD transgenic flies and overexpress this gene under the control of a ubiquitous Ga14 proteins ActGa14. Our study displayed that HmgD overexpression mutant died as late third instar larvae and malformed pupae during the larvae-prepupa transition,suggests that overexpression of HmgD has effects rather on the third instar larvae stage than on the first and second instar larvae from the mutant could not pupate.Ecdysone feeding experiments could not rescue these mutant larvae.We inferred that overexpression of HmgD did not affect the decrease in the ecdysone titer,but affected the transcription of some genes on the ecdysone signaling pathway.To detected the temporal patterns of expression for the Ecdysone Receptor gene EcR,extracted RNA from larvae samples of mid-third instar,late-third instar and prior to pupation,was analysed by RT-PCR.Our results showed that compare to wild type,the temporal expression patterns of EcR-A,EcR-C was not affected,while the expression of EcR-B was not detected in the mutant larvae.This result inferred that the overexpression of HMGD could probably block the transcription of EcR-B gene,which leading the failure of pupation.The phenotype of HmgD overexpression mutant in our study was the same as the EcR-B defection mutant,which confirmed our predict that ubiquitous overexpression of HMGD results in the failure of pupation by repressing the transcription of EcR-B and then induced the expression of some downstream genes like E75 on the ecdysone signaling pathway disorderly.Our previous studies have shown that ubiquitous overexpression of HmgD caused hemocytes proliferation and the formation of melanotic tumors in the Drosophila larvae. To clarify the mechanism of melanotic tumors appearance,we also generated RT-PCR analysis to test the transcriptional changes of PPAE and Spn27A,which encode the key factors in the phenoloxidase(PO)cascade pathway from larvae samples of mid-third instar,late-third instar and prior to pupation relative to wild type flies.Our result showed that in the HmgD overexpression mid-third instar larvae,the expression level.of Spn27A was lower than wild type,otherwise the expression level of PPAE was higher than wild type.It suggests that overexpression of HmgD affects the PO cascade pathway.Our explaination is as follow:activity of PPAE could not be inhibited,completely by a small quantity of Spn27A,which catalyze the inactive form PPO to the active form PO,caused the melanotic tumor appearance.We concluded that HMGD perhaps involve in the innate immunity of Drosophila, which is consistant with the result from vertebrate that the high concentrations in the inflammatory cell.We also found that ISWI,which encodes the nucleosome remodeling factor(NURF)catalyze subunit,and the gene cyclinA,cyclinE were overexpression in the HmgD overexpression mutant larvae.These results suggest that overexpression of HmgD causes hemocytes proliferation by multi- signals pathway and also demonstrates thai HmgDmay play a key role on the development of Drosophila.