Fertility test data for the triple knockout genotypes removing the three tandem STE24 paralogs. A fertility defect reveals itself only when males are homozygous for the triple knockout. “n” refers to the number of times a particular cross was set up. For each cross, five virgin males and females were mated to each other, and the crosses were turned into fresh vials every three to five days. Scoring of adult progeny was stopped after three vials. In all cases, where homozygous triple knockout male parents were involved, no progeny were produced past the second vial and usually not past the first vial. Df refers to Df(2R)Exel6065, which removes the three CG9000 genes and 37 additional genes.

Maximum likelihood phylogeny for CG9000 and paralogs. CG numbers for each gene denote the clades of Drosophila species for the respective locus. The figures above the nodes are the results from bootstrap analysis with 500 replicates (only bootstrap values ≥ 95 are shown). The scale is given as substitutions per site. (See Figure S6 for a similar analysis that includes CG30461.)

Dissected testes from age- and condition-matched male siblings from fertility crosses (see Figure 2). DAPI stains DNA and phalloidin-FITC was used to highlight actin structures. (A–C) Triple knockout heterozygotes, 3–5 days old. (D–F) Triple knockout homozygotes, 3 to 5 days old. (G–I) Triple knockout heterozygotes 10–16 days old. (J–L) Triple knockout homozygotes 10–16 days old. ac, actin cone (comprising the individualization complex); ag, accessory gland; sv, seminal vesicle; tb, testis base. Scale bar: 200 µM.

CG9000 genomic region and crossing scheme for targeted deletion of the tandem Drosophila STE24 paralogs. (A) Diagram depicting genomic organization of the CG9000 region, including upstream and downstream genes (not to scale). The three CG9000 paralogs (CG9000, CG9001, and CG9002) are shaded blue. The arrows with diagonal bends indicate the location of primers that were used in the design of the donor construct for gene deletion. The distances in base pairs below each primer position indicate the length of each flank that was cloned into the donor construct. Right-angled arrows indicate promoter regions for each gene. Inverted triangles indicate the location of P element or PiggyBac insertions. Note that CG30461 is indicated as dicistronic with CG9002, based on evidence from an fused cDNA from the Drosophila Genome Project (DGC:AT28654). (B) Crossing scheme showing the three generations required to isolate a targeted deletion of the CG9000 region. FLP:Flippase (a yeast recombinase); I-Sce-1: a restriction endonuclease; Pin is a dominant marker on the second chromosome; CyO, In(2LR)Cy and TM3 are balancer chromosomes. P{Donor} refers to the donor construct pRK2 into which the regions flanking the CG9000 cluster were cloned (see Materials and Methods). Note that this construct is marked with an asterisk (*) in the third generation, indicating movement from the third chromosome to the second. The use of balancer and Y chromosomes with the apoptosis promoter HID driven by heat shock maximizes selection of the correct intermediate genotypes. In addition, the donor construct includes another apoptosis promoter REAPER driven by GAL4 in the mapping generation that minimizes selection of false positives (Huang et al. 2008).

The fertility defect in triple knockout males is age dependent. Each data point represents the number of progeny resulting from a cross between five virgin homozygous males and females. The virgin male ages were as indicated (after eclosion) before exposure to the females. Fertility drops off to zero after about a week. Wild-type controls were set up for only three data points.

No changes in the principal stages of spermatogenesis in triple knockout vs. control genotypes. Phase contrast images of testes dissected from age- and condition-matched heterozygous or wild-type (A, C, E, G) and homozygous triple knockout (B, D, F, H) males. (A, B) Apical sections of testes from 10- to 15-day-old males, showing gonial and mitotic spermatogonia, elongated spermatids, and “waste bags,” spherical structures containing excess cytoplasm squeezed out of the spermatids during elongation. (C, D) Midsections of testes from 21-day-old males, showing meiotic spermatids, postmeitoic (round, phase light) spermatids, elongating spermatids, and onion stage mitochondria (nebenkern, phase dark). (E, F) Dissections releasing mature sperm from 3-day-old males. Note lesser amounts of sperm from triple knockout males. (G, H) Dissections releasing mature sperm from 8- to 10-day-old males. Note that although there is still less sperm in the triple knockout homozygote (H) than in the wild-type (G), at this point the triple knockouts are virtually completely sterile. Scale bars: 100 µM (A, B, E, F, G, H) and 50 µM (C, D).

Individualization complexes form normally in triple knockout flies, but elongated spermatids exhibit a cytoplasmic defect. Fluorescence images for heterozygous and homozygous triple knockout spermatids. All males had been mated and were between three and four weeks old. (A, B) Low-power images (20X; scale bar, 50 µM) showing Don Juan–GFP localization in the proximal region of the testes and seminal vesicles. Note that the Don Juan–GFP triple knockout stock was built over a reciprocal translocation (apterous^Xa) between the second and third chromosomes (to minimize balancer breakdown), which means that the homozygous triple knockout males have in fact a double dose of Don Juan–GFP relative to their siblings, yet the heterozygotes consistently fluoresced much more brightly under identical camera settings, supporting the conclusion that far less mature sperm is transported into the seminal vesicles in the triple knockout (see also Figure S10). (C, D) Actin-based individualization complexes (scale bar, 20 µM) are labeled with phalloidin-TRITC, and sperm flagella are labeled with Don Juan–GFP. Although the individualization complex looks normal in both genotypes, the triple knockout exhibits a grainier appearance of GFP. This grainier appearance is more obvious in GFP-only images for the same genotypes: E (scale bar, 200 µM) and E′ (scale bar, 20 µM) are from heterozygous males; F and F′ (same scaling) are from homozygous triple knockout males. sv, seminal vesicle; tb, testes base.