| Adult Stem Cells(ASCs)are a kind of cells in adult tissues that have the ability to self-renew and differentiate filial cells to maintain the homeostasis of the tissue.The homeostasis can be regulated not only by endogenous factors of stem cells,but also by exogenous factors in the niche of stem cells.Without stem cells,tissue will degrade eventually due to the finite life span of differentiated adult cells.Germline Stem Cells(GSCs),as a member of ASCs,play a very significant role,which is related to the reproduction of race.Drosophila melanogaster is a classic model,and its ovary has become one of the best model systems for the study of GSCs.Hsc70-4 gene belongs to the family of heat shock protein 70,and encode heat shock protein Hsc70-4.The research has shown that Hsc70-4 heat shock protein involved in regulation of various biological processes,such as cell response to the stimulus,partner proteins mediated protein folding,response to the topology of incorrect protein,regulate the expression of gene transcription and secretion of the neurotransmitter,vesicle mediated transport,etc.Through previous studies,we found that knockdown of Hsc70-4 gene in Drosophila ovaries resulted in abnormal ovarian morphology,and further investigation found that the amount of GSCs in the ovary with abnormal morphology was seriously reduced.Therefore,we designed a series of experiments to reveal that how Hsc70-4 regulates the fate of Drosophila GSCs.Firstly,the expression level of Hsc70-4 gene in various tissues of Drosophila was investigated by real-time quantitative PCR(q PCR).Whole body,head,intestine,martensii tube,fat body,muscle,ovary and testis of wild-type Drosophila Oregon were eight organs that selected as the experimental subjects.The results showed that Hsc70-4 gene was specifically expressed in various tissues of Drosophila melanogaster,and the expression of Hsc70-4 gene was at a high level in Drosophila ovary.In order to investigate whether the Hsc70-4 gene has affected the development of Drosophila ovary,two groups of Hsc70-4 RNA interfering(RNAi)strain that we ordered are:P{trip.hmj21529}att P40 and P{y[+T7.7]V[+T1.8]=trip.glv21049}att P2 and the Hsc70-4 gene expression in Drosophila ovary is regulated respectively by UAS-Gal4system.The results showed that the wild-type Drosophila ovary was full,with normal ovary tube and embryos,while the two groups of Drosophila ovary with knockdown Hsc70-4 gene expression showed abnormal ovary size,atrophy of ovary tube and no embryos in ovary.The phenomena preliminarily suggest that Hsc70-4 gene has influence on the development of ovary.To figure out how Hsc70-4 genes affect the development of ovary,two groups of Hsc70-4 RNAi strains respectively hybridized with strain of endogenous Drosophila(Nos P-GVP)and strain of exogenous Drosophila(c587-gal4)through the genetic hybridization and finally we obtained the Drosophila with endogenous or exogenous knockdown of Hsc70-4 gene.Using immunohistochemical techniques to explore that with different number of days after eclosion(AE)their number of changes of GSCs in ovaries.The results showed that,as control groups,wild-type Drosophila and two groups of Hsc70-4 RNAi strain(not the knockdown group),which ratios of normal germarium in 14days after eclosion is over 95%.Two groups Drosophila of endogenous knockdown of Hsc70-4 at day 1 AE have reduced GSCs seriously and the ratio of normal germarium is 0.These results indicate that endogenous knocking down Hsc70-4 gene would seriously affect the ability to maintain GSCs in Drosophila ovary.However,at day 14 AE,ratios of normal germarium in the two groups of Drosophila with exogenous knockdown of Hsc70-4 gene was 94.2%and 98.4%,which was no different from the data in the control group,indicating that the exogenous knockdown of Hsc70-4 gene did not affect the maintenance of GSCs in Drosophila ovary.All these results indicate the possibility of endogenous regulation of Hsc70-4 gene in the fate of ovary germline stem cells.To confirm two groups of Drosophila strains with endogenous knockdown Hsc70-4genes,their Hsc70-4 gene expression was positively correlated with the maintenance of GSCs in their ovaries.Using q PCR,the relative expression of Hsc70-4 gene in the ovary of two groups of endogenously knockdown Hsc70-4 gene Drosophila strain was detected from the m RNA level.The q PCR dated showed that the expression of Hsc70-4 gene in the two endogenous knockdown groups decreased by 1.4 times and 2.6 times,respectively,compared with the wild type,both of which were significantly reduced.The results showed that the down-regulation of endogenous expression of Hsc70-4 genes in Drosophila ovary is closely related to the inability of germline stem cells to maintain.For further evidence that Hsc70-4 gene endogenously regulate the fate of germline stem cells,GSCs clones of endogenous mutations in Hsc70-4 genes induced by FLP/FRT mitotic recombination,Statistics 1d?7d?14d the number of GSCs cloning numbers labeled with GFP negative.The results showed that:14 days later,the percentage of GSCs clones that were GFP-negatively labeled with FRT82B in the control group was basically the same as the first day at 41.9%,while the percentage of GSCs markers of Hsc70-4 mutant cells in the two experimental groups(FRT82B,Hsc70-4[L3929]and FRT82B,Hsc70-4[03550])showed a decreasing trend,Hsc70-4[L3929]experimental group decreased by 20.7%after 14days,in the FRT82B,Hsc70-4[03550]experimental group,the number of GSCs markers was even more seriously lost.After 14 days,the GSCs percentage of the labeled was only5.2%.The results show that endogenous deletion of Hsc70-4 gene can also cause the loss of GSCs,which proves that Hsc70-4 gene plays an endogenous role in regulating the maintenance of germline stem cells in Drosophila ovary.To get rid of the genetic background and verify the function of Hsc70-4 genes,a series of Hsc70-4 transgenic vectors were constructed in this project,and then related the Hsc70-4 genetically modified Drosophila.Supplement the Hsc70-4 gene in the context of mutations to rescue the GSCs loss phenotype caused by endogenous knockdown or mutation of the Hsc70-4 gene.Hsc70-4 gene endogenous high knockdown phenotypes were rescued from three aspects:systemic(Hsc70-4P-Hsc70-4 transgenic Drosophila),endogenous(nos P-Hsc70-4 transgenic Drosophila)rescued and induced endogenous(UASP-Hsc70-4 transgenic Drosophila),we performed the rescue assay in Hsc70-4[L3929]/Hsc70-4[03550]mutant by using the transgenic Drosophila nos P-Hsc70-4.The results show that Drosophila caused by endogenous knockdown of the Hsc70-4 gene had no GSCs phenotype in the germarium,and part of the GSCs was restored after the rescue,and the proportion of GSCs marked by the endogenous mutation of the Hsc70-4 gene was also return to normal levels.In conclusion,the above results demonstrate that Hsc70-4 genes regulate the fate of germline stem cell.Next,using Hsc70-4P-GFP transgenic Drosophila to verify the expression pattern of the Hsc70-4 gene in the ovary of Drosophila,and observe the expression site of Hsc70-4gene in Drosophila ovary by immunostaining.The results showed that the Hsc70-4 gene was expressed in both the endogenous and exogenous environment of the Drosophila ovary.This experimental result confirms the veracity of the conclusion that the Hsc70-4gene endogenously regulates the fate determination of germline stem cells.We have known that knockdown or mutation of Hsc70-4 gene can cause the loss of GSCs number.So,is the loss of GSCs due to differentiation?In order to verify this hypothesis,we overexpressed the Hsc70-4 gene in the ovary,and observed whether it will delay the differentiation of GSCs and Cystoblasts.From endogenous(nos P-Hsc70-4/nos P--Hsc70-4 genotype Drosophila),induced endogenous(UASP-Hsc70-4/UASP-Hsc70-4;Nos-GVP/Nos-GVP genotype Drosophila)and heat shock(hs P-Hsc70-4/hs P-Hsc70-4genotype Drosophila),overexpressing the Hsc70-4 gene and counting the number of“spectrosome”(the sum of the number of GSCs and CBs)in the germarium of the Drosophila ovary.The results showed that the average number of"spectrosome"in the three overexpression groups was 6.90,6.49,and 6.30,which was a significant increase compared with the average number of wild-type Oregon"spectrosome"3.28.The above results indicate that overexpression of Hsc70-4 gene can effectively delay the differentiation of GSCs and CBs,the loss of GSCs caused by knockdown or mutation of Hsc70-4 gene may be due to the differentiation of GSCs.Based on the above research results,it is shown that the Hsc70-4 gene is specifically expressed in Drosophila tissues and both endogenously and exogenously in the Drosophila ovary,but the Hsc70-4 gene only endogenously regulates the fate of germ stem cells of Drosophila ovary.The knockdown or mutation of Hsc70-4 gene will lead to the failure of GSCs self-renewal and imbalance of differentiation,which will make GSCs unable to maintain and advance to differentiation. |
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