Drosophila Rbp6 is an orthologue of vertebrate Msi-1 and Msi-2, but does not function redundantly with dMsi to regulate germline stem cell behaviour.

The vertebrate RNA-binding proteins, Musashi-1 (Msi-1) and Musashi-2 (Msi-2) are expressed in multiple stem cell populations. A role for Musashi proteins in preventing stem cell differentiation has been suggested from genetic analysis of the Drosophila family member, dMsi, and both vertebrate Msi proteins function co-operatively to regulate neural stem cell behaviour. Here we have identified a second Drosophila Msi family member, Rbp6, which shares more amino acid identity with vertebrate Msi-1 and Msi-2 than dMsi. We generated an antibody that detects most Rbp6 splice isoforms and show that Rbp6 is expressed in multiple tissues throughout development. However, Rbp6 deletion mutants generated in this study are viable and fertile, and show only minor defects. We used Drosophila spermatogonial germline stem cells (GSC's) as a model to test whether Drosophila Msi proteins function redundantly to regulate stem cell behaviour. However, like vertebrate Msi-1 and Msi-2, Rbp6 and Msi do not appear to be co-expressed in spermatogenic GSC's and do not function co-operatively in the regulation of GSC maintenance. Thus while two Msi family members are present in Drosophila, the function of the family members have diverged.

Dmp53 is sequestered to nuclear bodies in spermatogonia of Drosophila melanogaster.

p53 family members have been implicated in regulation of genomic integrity and apoptosis in a variety of tissues. The Drosophila family member, Dmp53, primarily functions to regulate apoptosis in developing and regenerating tissues but loss of function mutants are viable and fertile. Dmp53 exhibits a striking expression pattern in the male germline with high levels found in nuclear bodies in pre-meiotic germ cells. The localisation of Dmp53 to nuclear bodies is dependent upon Dmp53 complexes being able to bind DNA, and although dmp53 mutants do not affect germline stem cell (GSC) maintenance or differentiation, GSCs are sensitive to overexpression of Dmp53 but maturing spermatogonia are not. Dmp53 thus has differential effects depending upon the stage of male germline maturation.

GAL4 enhancer traps that can be used to drive gene expression in developing Drosophila spermatocytes.

The Drosophila testis has proven to be a valuable model organ for investigation of germline stem cell (GSC) maintenance and differentiation as well as elucidation of the genetic programs that regulate differentiation of daughter spermatogonia. Development of germ cell specific GAL4 driver transgenes has facilitated investigation of gene function in GSCs and spermatogonia but specific GAL4 tools are not available for analysis of postmitotic spermatogonial differentiation into spermatocytes. We have screened publically available pGT1 strains, a GAL4-encoding gene trap collection, to identify lines that can drive gene expression in late spermatogonia and early spermatocytes. While we were unable to identify any germline-specific drivers, we did identify an insertion in the chiffon locus, which drove expression specifically in early spermatocytes within the germline along with the somatic cyst cells of the testis.

Differential roles of HOW in male and female Drosophila germline differentiation.

The adult gonads in both male and female Drosophila melanogaster produce gametes that originate from a regenerative pool of germline stem cells (GSCs). The differentiation programme that produces gametes must be co-ordinated with GSC maintenance and proliferation in order to regulate tissue regeneration. The HOW RNA-binding protein has been shown to maintain mitotic progression of male GSCs and their daughters by maintenance of Cyclin B expression as well as suppressing accumulation of the differentiation factor Bam. Loss of HOW function in the male germline results in loss of GSCs due to a delay in G2 and subsequent apoptosis. Here we show that female how mutant GSCs do not have any cell cycle defects although HOW continues to bind bam mRNA and suppress Bam expression. The role of HOW in suppressing germ cell Bam expression appears to be conserved between sexes, leading to different cellular outcomes in how mutants due to the different functions of Bam. In addition the role in maintaining Cyclin B expression has not been conserved so female how GSCs differentiate rather than arrest.

HOW is required for stem cell maintenance in the Drosophila testis and for the onset of transit-amplifying divisions.

The mechanisms by which germline stem cells (GSCs) in the Drosophila testis undergo asymmetric division to regenerate a stem cell as well as a daughter (gonialblast) that will only undergo a further four mitotic divisions prior to entering premeiotic S phase and differentiating into a cyst of spermatocytes are not fully resolved. Here we demonstrate that the HOW RNA-binding protein is required for maintenance of CycB and therefore mitotic progression in GSCs and gonialblasts as well as determining the timing of the spermatogonial divisions. HOW is normally expressed in a complementary pattern to Bam in the germline and bam mRNA is bound by HOW in vivo. Ectopic expression of the HOW(L) isoform is associated with a delay in accumulation of Bam to the level required for differentiation, resulting in extra mitotic divisions. Spatiotemporal regulation of HOW expression is therefore required to specify the four spermatogonial transit-amplifying divisions.