Studies On Hsp83 Gene Regulating The Fate Of Drosophila Ovarian Germline Stem Cell

ASCs are characterized by self-renewal and potential differentiation,therefore how to keep a balance between self-renewal and differentiation is very crucial for tissue homeostasis.Previous Studies from various stem cell systems have shown that the intrinsic factors came from stem cells are necessary for this balance,meanwhile,the extrinsic signaling molecules from the microenvironment(also called “niche”)surrounding ASCs also control this balance.Germline Stem Cells(GSCs)is one type of ASCs,which play an important role in lots of life processes.As a classic model,The Drosophila ovary provides an exemplary model for the study the biology of stem cells and their niches.Drosophila hsp83 is a homolog of mammalian hsp90 encoding a heat shock protein Hsp83.Hsp83 protein regulates multiple physiological processes,such as morphogenesis,oogenesis,etc.In the process of screening the sterile flies,we found that the number of GSCs was reduced in the hsp83 mutation strain compared with the Oregon wild-type.Thus,we designed a series of experiments to reveal the mechanism of hsp83 gene regulating the maintenance of Drosophila GSCs.To confirm the function of hsp83 gene,we checked the GSCs number in three groups of hsp83 mutants(hsp8308445-/-,hsp8308445/hsp83e6 d,hsp8308445/hsp83e6a)in different days(1d,7d,14 d,21d)after eclosion(AE)with immunohistochemistry technique.The results show that the germarium of wild-type usually contains 2-3 GSCs.The percentages of normal germaria in three groups of hsp83 mutants at day 1 AE are respectively 98.77%,100% and 80.43%,while the percentages are decreased to 76.90%,73.68% and 34.23% at day 21 AE respectively.Among them,the mutant of hsp8308445/hsp83e6 a possesses the most severe phenotype,and about 44% of germaria have no GSCs.Next,we detected the number of GSCs in knockdown of hsp83 flies in different days(1d,7d,14d)after eclosion.The results show that the ratios of normal germaria in the control are 98.36%,94.64% and 92.31%,respectively.When hsp83 is knocked down in the GSCs,the proportions of normal germaria are decreased to 63.69%,53.56%,and 12.18%,respectively.Meanwhile,when hsp83 is knocked down in the niche,the ratios of normal germaria are respectively decreased to 92.47%,80.49% and 69.89%.To verify the results,we performed a rescue assay in the hsp83 mutant(hsp8308445/hsp83e6 d,hsp8308445/hsp83e6a)by using transgenic fly hsp83P-hsp83.The results show that the percentages of normal germaria at day 21 AE are 90.79% and 89.78%,and recover to normal level.These results suggest that hsp83 regulates the maintance of Drosophila ovarian GSCs.The above data have shown that the intrinsically reduced of hsp83 significantly affects the maintenance of GSCs number.To validate this result,we exerted mitotic recombination technique to analyze the fate of mutant GSCs.The rate of marked GSCs clones from FRT control decreased weakly from the initial of 51.04% at day 2 to the final of 48.54% at day 20 after heat-shock treatments(AHST).This manifests that only 4.9% of the marked GSCs are lost away during the 20-day AHST.By contrast,under the same experimental conditions,the initial marked hsp8308445,hsp83e6 d and hsp83e6 a mutant GSC clones are respectively measured as 51.52%,47.37% and 46.45% at day 2 AHST,where as the marked hsp8308445,hsp83e6 d and hsp83e6 a mutant GSCs declined to the ratios of 32.71%,24.29% and 2.90% respectively at day 20 AHST.Among them,about 93.8% of labelled GSCs with hsp83e6 a mutation are loss away.This result supports the point that lack of hsp83 intrinsically results in GSCs loss.To determine whether the hsp83 gene is endogenous or exogenous factor that regulates the fate of GSCs,we designed a series of intrinsic and extrinsic rescue assays.Firstly,we performed the rescue assay in hsp8308445/hsp83e6 a mutant by using the transgenic fly NosP-hsp83.The result shows that the percentage of normal germaria is 35.07%,which has no significant difference compared to the hsp8308445/hsp83e6 a mutant at day 21 AE with the percentage of 33.74%.The result shows that only endogenous supplementation of hsp83 failed to rescue the GSCs deficient phenotype.In addition,similar result is obtained by endogenous rescue assay by combining the UASP-hsp83 transgenic fly with NosP-gal4 line.Secondly,the result of exogenousrescue assay using the UASP-hsp83 transgenic fly and c587-gal4 line shows that the ratio of normal germaria has no significant difference with hsp83 mutant at day 21 AE.The result suggests that only exogenous supplementation of hsp83 also failed to rescue the GSCs loss phenotype.Taken together,these data strongly indicate that hsp83 functions both as an intrinsic and as an extrinsic factor for regulating GSCs fate.To reveal the relationship between hsp83 and BMP signaling pathway,we investigated the genetic relationship between hsp83 and Dad&bam.Firstly,we examined Dad and bam expression pattern in hsp83 mutant background by using Dad-GFP and bam-GFP.The results show that there is no big difference between the control and hsp83 mutant fly.Secondly,we counted the number of GSCs in gene single mutation fly(SMF)of bam/hsp83 and hsp83&bam double mutation fly(DMF).The results show that the germarium of bam SMF contains many GSC-like cells.The percentages of normal germaria in hsp83 SMF in four points(1d,7d,14 d,21d)are respectively 75%,59.20%,47.46% and 34.23%,while the ratios of normal germaria in hsp83&bam DMF are 75.81%,30.77%,20.45% and 14.63%,respectively.The results indicate that the phenotype of hsp83&bam DMF is similar to that of the hsp83 SMF.Therefore,we conclude that hsp83 is parallel to the "BMP-bam" signaling pathway.Finally,we detected the apoptosis of hsp83 mutant GSCs by TUNEL technique,and there is no significant apoptosis in mutant GSCs compared with the control.Based on this,we hypothesized that overexpressed hsp83 in ovary maybe delay GSCs/GBs differentiation.To test the point,we performed hsp83 gene overexpression assay and measured the number of spectrosome-contained GSC/GBs(SCGs)in germaria.The results show that the average number of SCGs in wild-type is 3.75,and the average numbers of SCGs in flies(c587-gal4;UASP-hsp83;NosP-gvp and hsp83P-hsp83)are respectively 4.55 and 4.29,there are slight increases in SCGs number compared to the control.The average numbers of SCGs in flies(UASP-hsp83;NosP-gvp,NosP-hsp83,NosP-gal4;UASP-hsp83,UASP-hsp83;Act-gal4 and UASP-hsp83;Tub-gal4)are 4.01,3.97,3.85,3.92 and 3.88,respectively,there are no big difference between the control and hsp83 overexpression fly.Taken together,these data imply that the increased hsp83 activity could not efficiently delay GSC/GB differentiation.In conclusion,our findings show that Drosophila hsp83 gene play a key role in regulating the GSC fate by both an endogenous and an exogenous mechanism.Furhermore,hsp83 controls GSCs fate independently on the BMP-bam signaling pathway.