Investigating spermatogenesis in Drosophila melanogaster

Abstract

The process of spermatogenesis in Drosophila melanogaster provides a powerful model system to probe a variety of developmental and cell biological questions, such as the characterization of mechanisms that regulate stem cell behavior, cytokinesis, meiosis, and mitochondrial dynamics. Classical genetic approaches, together with binary expression systems, FRT-mediated recombination, and novel imaging systems to capture single cell behavior, are rapidly expanding our knowledge of the molecular mechanisms regulating all aspects of spermatogenesis. This methods chapter provides a detailed description of the system, a review of key questions that have been addressed or remain unanswered thus far, and an introduction to tools and techniques available to probe each stage of spermatogenesis.

Keywords: Cytokinesis; Drosophila; Germ line; Meiosis; Spermatogenesis; Stem cell.

Figure 1. Spermatogenesis in Drosophila melanogaster

A) Schematic of spermatogenesis. Germline stem cells (GSC, light green) and somatic cyst stem cells (CySC, light grey) contact hub cells (red). GSCs divide to self-renew and generate a daughter gonialblast (GB). GBs undergo 4 rounds of mitosis with incomplete cytokinesis to generate a cyst of 16 spermatogonia. Spermatogonial cysts (dark green) are surrounded by early cyst cells (dark grey). Spermatogonia initiate terminal differentiation as spermatocytes, which undergo 2 meiotic divisions with incomplete cytokinesis to generate 64 haploid spermatids. B) Phase contrast micrograph of a wild type testis. Asterisk denotes apical tip.

Figure 2. Immunofluorescence images of testis tips highlighting different tools used study early steps in spermatogenesis

A) Immunofluorescence (IF) micrograph depicting GSCs and early cyst cells surrounding the apical hub (asterisk). Stat92E protein (green) is stabilized in GSCs, and early cyst cells (including CySCs) express the transcription factor Traffic Jam (red). B) IF image showing the hub marked by antibody staining for the cell surface marker Fasciclin3 (Fas3; red) and spermatocytes expressing Sa:GFP (arrow). Brackets denote the transit-amplification zone recognizable by dense DAPI staining of DNA in spermatogonia. C) IF image showing the progression of germline development as marked by the extension of a-spectrin⁺ fusomes in interconnected spermatogonia and spermatocytes. Germ cells are stained with an antibody to the RNA helicase Vasa (green). Asterisk denotes the hub.

Figure 3. Use of FRT-mediated recombination strategies in Drosophila melanogaster

A) FRT (flippase recognition target) sites located on the same chromosome permits generation of ‘flip-out’ clones. Upon expression of Flippase (FLP), recombination occurs, removing intervening sequences and permitting expression of ^Gal4. Resultant “clones” will be marked by the expression of the GAL4-responding transgene (i.e., UAS-GFP). If recombination occurs in a stem cell, progeny of that stem cell will be similarly marked. This strategy does not require mitosis. B) Use of the MARCM system permits the generation of clones and lineage-tracing studies. As shown above, upon expression of FLP in mitotic cells, FRT sites from homologous chromosomes, one of which contains a specific mutation, will recombine in a sitespecific manner. Wild type chromosomes also carry the GAL80 repressor, which represses GAL4 activity such that expression of the GAL4-responding transgene (UAS-GFP) is suppressed. Upon loss of GAL80, cells containing chromosomes carrying mutations will be marked by the expression of GFP.