Nucleoporin98-96 Function Is Required for Transit Amplification Divisions in the Germ Line of Drosophila melanogaster

Production of specialized cells from precursors depends on a tightly regulated sequence of proliferation and differentiation steps. In the gonad of Drosophila melanogaster, the daughters of germ line stem cells (GSC) go through precisely four rounds of transit amplification divisions to produce clusters of 16 interconnected germ line cells before entering a stereotypic differentiation cascade. Here we show that animals harbouring a transposon insertion in the center of the complex nucleoporin98-96 (nup98-96) locus had severe defects in the early steps of this developmental program, ultimately leading to germ cell loss and sterility. A phenotypic analysis indicated that flies carrying the transposon insertion, designated nup98-962288, had dramatically reduced numbers of germ line cells. In contrast to controls, mutant testes contained many solitary germ line cells that had committed to differentiation as well as abnormally small clusters of two, four or eight differentiating germ line cells. This indicates that mutant GSCs rather differentiated than self-renewed, and that these GSCs and their daughters initiated the differentiation cascade after zero, or less than four rounds of amplification divisions. This phenotype remained unaffected by hyper-activation of signalling pathways that normally result in excessive proliferation of GSCs and their daughters. Expression of wildtype nup98-96 specifically in the germ line cells of mutant animals fully restored development of the GSC lineage, demonstrating that the effect of the mutation is cell-autonomous. Nucleoporins are the structural components of the nucleopore and have also been implicated in transcriptional regulation of specific target genes. The nuclear envelopes of germ cells and general nucleocytoplasmic transport in nup98-96 mutant animals appeared normal, leading us to propose that Drosophila nup98-96 mediates the transport or transcription of targets required for the developmental timing between amplification and differentiation

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

Drosophila Dynein Intermediate Chain Gene, Dic61B, Is Required for Spermatogenesis

This study reports the identification and characterization of a novel gene, Dic61B, required for male fertility in Drosophila. Complementation mapping of a novel male sterile mutation, ms21, isolated in our lab revealed it to be allelic to CG7051 at 61B1 cytogenetic region, since two piggyBac insertion alleles, CG7051c05439 and CG7051f07138 failed to complement. CG7051 putatively encodes a Dynein intermediate chain. All three mutants, ms21, CG7051c05439 and CG7051f07138, exhibited absolute recessive male sterility with abnormally coiled sperm axonemes causing faulty sperm individualization as revealed by Phalloidin staining in Don Juan-GFP background. Sequencing of PCR amplicons uncovered two point mutations in ms21 allele and confirmed the piggyBac insertions in CG7051c05439 and CG7051f07138 alleles to be in 5′UTR and 4th exon of CG7051 respectively, excision of which reverted the male sterility. In situ hybridization to polytene chromosomes demonstrated CG7051 to be a single copy gene. RT-PCR of testis RNA revealed defective splicing of the CG7051 transcripts in mutants. Interestingly, expression of cytoplasmic dynein intermediate chain, α, β, γ tubulins and α-spectrin was normal in mutants while ultra structural studies revealed defects in the assembly of sperm axonemes. Bioinformatics further highlighted the homology of CG7051 to axonemal dynein intermediate chain of various organisms, including DNAI1 of humans, mutations in which lead to male sterility due to immotile sperms. Based on these observations we conclude that CG7051 encodes a novel axonemal dynein intermediate chain essential for male fertility in Drosophila and rename it as Dic61B. This is the first axonemal Dic gene of Drosophila to be characterized at molecular level and shown to be required for spermatogenesis

Dynein light chain 1 functions in somatic cyst cells regulate spermatogonial divisions in Drosophila

Stem cell progeny often undergo transit amplifying divisions before differentiation. In Drosophila, a spermatogonial precursor divides four times within an enclosure formed by two somatic-origin cyst cells, before differentiating into spermatocytes. Although germline and cyst cell-intrinsic factors are known to regulate these divisions, the mechanistic details are unclear. Here, we show that loss of dynein-light-chain-1 (DDLC1/LC8) in the cyst cells eliminates bag-of-marbles (bam) expression in spermatogonia, causing gonial cell hyperplasia in Drosophila testis. The phenotype is dominantly enhanced by Dhc64C (cytoplasmic Dynein) and didum (Myosin V) loss-of-function alleles. Loss of DDLC1 or Myosin V in the cyst cells also affects their differentiation. Furthermore, cyst cell-specific loss of ddlc1 disrupts Armadillo, DE-cadherin and Integrin-βPS localizations in the cyst. Together, these results suggest that Dynein and Myosin V activities, and independent DDLC1 functions in the cyst cells organize the somatic microenvironment that regulates spermatogonial proliferation and differentiation