| Stem cells are a group of cells possessing two properties,self-renewal and the potential to differentiation,which leads to their broad applications in regeneration medicine.The studies on the fate determination of germline stem cells（GSCs）always is a charming field in stem cell biology.Drosophila melanogaster is a good model organism in developmental biology for its powerful genetic tools.The Drosophila ovarian GSC provides an excellent research system for the reliable stem cell markers and clear germ cell lineage.The Lamin protein,as a kind of intermediate filament（IF）proteins,provides skeletal support for the nuclear membrane.Meantime,Lamin is also involved in these biological processes such as chromatin organization,DNA replication and DNA damage repair.Our previous work found that the number of ovarian GSCs in the lamin mutant flies decreased with time.To confirm the finding and explore the mechanism of lamin regulating the GSC fat,we performed the following experiments:Firstly,we statistically analyzed the GSC number of the lamin mutant and wild type ovaries collected at days 1,and 14 post-eclosion by immunostaining GSCs.We found that the ratios of normal germaria（generally containing 2-3 GSCs）examined at the two time-points were 97.7%and 96.4%,respectively.The data suggest there is no big difference in GSC number at different time.In contrast,the proportions of the three lamin mutants(lamA25/lamA25、lamA25/lamK2、lamA25/lam046434643 and lamK2/lam04643)checked at day 1 were 78.7%,81.8%,84.8%and 88.0%,respectively.However,the ratios were counted as 27.9%,36.2%,37.6%and 48.1%,respectively.The data show that the number of normal germaria decreased significantly.The above results suggest that the lamin gene is required for the maintenance of Drosophila ovarian GSC.Secondly,we measured the ovarian GSC number in endogenous/exogenous lamin-RNA interference（RNAi）background by UAS/gal4 system,and the number of GSCs were counted at days 1,7 and 14d after eclosion.The data showed that the proportion of normal germaria sustained at high level,counted as 92.0%in two weeks,indicating that there was no change in the number of ovarian GSC.In contrast,the proportion of normal germaria from two lamin RNAi ovaries measured at day 1 were84.1%and 72.1%,respectively.After 14 days,the ratios were 31.2%and 36.6%,respectively.The results showed that endogenous knockdown of lamin resulted in a significant decrease in the numbers of normal germaria,suggesting a severe decline in the number of ovarian GSCs.Next,we did the lamin exogenous RNAi assay,the results showed that there was no apparent change between lamin-RNAi and the wild type control（P>0.05）.this result indicates that the exogenous knockdown of lamin does not affect GSC maintenance.In addition,GSC mutant clones were induced by mitotic recombination technique,and the numbers of labeled GSC clones were counted at day 1,7 and 14.The results showed that the percentage of GSC（control:FRT40A）slightly decreased from 43.7%at day 1 to 42.1%at day 14.While the percentage of GSCs from lamin mutant clones(FRT40A,lamA25、FRT40A,lamK2 and FRT40A,lam04643)at three experimental time-points decreased dramatically from43.8%,45.0%and 44.0%at day 1,to 15.1%,16.7%and 17.3%at day 14.The data showed that there were 65.5%,62.9%and 60.7%of GSCs were lost in two weeks,respectively.Taken together,these results suggest that the lamin gene plays an endogenous role in regulating GSC maintenance in Drosophila ovary.To furtherly validate the lamin gene’s function,we constructed a series of lamin transgenic vectors to rescue the phenotype caused by the Lamin-knockdown.The results showed that endogenous supplementation of lamin gene can fully rescue the phenotype of lamin mutation.Only exogenously supplied lamin fail to rescue the GSC loss phenotype,indicating that the lamin endogenously plays a role in maintaining Drosophila ovarian GSC.In addition,the expression pattern of Lamin protein was detected by immunohistochemical staining technique,we found that Lamin protein is highly expressed in ovarian GSCs and localized to the nuclear membrane,indicating that lamin plays an important role in GSC.To substantiate the relationship between lamin gene expression and GSC maintenance,we detected the lamin mRNA level with real-time PCR（qPCR）.The results showed that,compared to wild type,the lamin expression level were significantly reduced both in the mutant and in RNAi ovaries.In addition,we also measured the expression of Lamin protein by Western blot.The results showed that the Lamin protein expression level were dramatically decreased.All of data consistently suggest that the down-regulation of lamin gene is responsible for the loss of GSCs in Drosophila ovary.Subsequently,we investigated the genetic relationship between lamin and the well-known BMP/Dpp-bam signaling pathway using the bamP-GFP reporter.The ratio of GFP-positive GSCs from the control was accounted as 0.3%,indicating that the bam silencing is necessary for GSC maintenance.In the lamin mutant ovaries,The ratios of GFP-positive GSCs from three lamin transheterozygotes(bamP-GFP;lamA25/lamA25、bamP-GFP;lamA25/lamK2 and bamP-GFP;lamK2/lam04643)were 18.8%,30.3%and 12.5%,respectively.The data indicates that the lamin gene is partially involved in the silence of the bam expression in Drosophila ovarian GSC（upstream of bam）.In other words,lamin mainly locates downstream of bam,or parallels the BMP/Dpp-bam signaling pathway to maintain stem cells.Since the lamin mutation caused the loss of GSCs,but where did the lost GSCs go?To answer this question,TUNEL technology was performed to detect the apoptosis in lamin mutant GSCs.The results showed that the incidence of GSC apoptosis was counted as 1.8%in wild-type Drosophila ovary,and the two lamin mutant GSCs were 5.1%and 5.3%,respectively.The data suggest that no apparent increases of GSCs apoptosis rate in the lamin mutant background.In addition,no increased apoptotic rate was found in the endogenous lamin mutant GSC obtained by mitotic recombination.Put together,the results imply that the mutation of lamin leads to GSCs’pre-differentiation in Drosophila ovary.Our work has deepened the understanding of the mechanisms of GSC fate determination. |
