Studies On Gilgamesh Regulating The Maintenance Of Germline Stem Cells In Drosophila Testis

Stem cell is responsible for multiple physiological processes such as embryonic development,blood cells regeneration,tissues repair and so on.Stem cells are characterized as the self-renewal and differentiation.Germline stem cells(GSCs)play an important role in the process of gametogenesis and maintaining of the homeostasis of the reproductive system.Drosophila testis provides an excellent model for searching for the factors which regulate the self-renewal and differentiation of GSCs.Drosophila gilgamesh(gish),a homologue of human CK1-?,which extensively expresses in glial neuron cells,germline cells and muscle cells and plays key roles in glial cell migration,membrane trafficking,spermatogenesis and olfactory learning.Here,we have fund that gish is required for maintenance of germline stem cells in Drosophila testis.Firstly,we perform the genetic screen of male sterile Drosophila lines to search for the candidate genes which may involved in the maintenance of the testis GSCs.The results show that the number of GSCs of gish mutant allele dramatically decreases with the elapse of time compared to the wild type of Oregon.To confirm this result,we conduct genetic crosses between different alleles of gish mutants and check the GSCs number in different days(1d,10 d,20d)by immunostaining the testis.The results show that the average GSCs numbers of three gish mutants of gishKG03891/gish04895,gishKG03891/gishMI08417,gish04895/gishMI08417 in three points are respectively 6.5,5.6,3.7,while the average numbers of GSCs in wild type are respectively 8.1,7.7,6.6.This results demonstrate that the mutation of gene leads to the loss of GSCs in Drosophila testis.Secondly,we design the primes to perform RT-PCR to clone the different ORF(open reading frame)of gish.Then conduct realtime PCR to check the transcriptional level of the gish transcripts.We generate the transgenic vector and do the microinjection of embryos.We totally gain 8 real sequences of gish ORF sequences in testis of Drosophila depend on the gene sequencing.The expression level of 6 gish genes(BEFJKI)are twenty-six times higher than that of 2 gish(AC).This result suggests that 6 transcripts of gish maybe play a key role in the maintenance of testis GSCs.The total expression level of three gish mutants of gishKG03891/gish04895,gishKG03891/gishMI08417,gish04895/gishMI08417 dramatically decrease compared to wild type by qPCR.We get gish transgenic strains by microinjecting P{Uasp-gishF} to the embryos of fly.Thirdly we perform the rescue assay by using the transgenic allele of P{NosP-gish F} and P{UASp-gishF}.The results show that the average GSCs numbers of three rescued lines in three points(day1,10,20)are respectively 7.0,7.0,5.8,the number of GSCs recoveries to the normal level.This result indicates gish regulates the maintenance of the GSCs in Drosophila testis.Finally,we detect the expression pattern of bam and dad in gish mutant background.The results show that it has no difference between wild type and gish mutant fly.This results suggest that gish functions downstream of bam & dad,or parallel of the Bmp pathway.