Author Contributions
Conceived and designed the experiments, K.G.M., K.R., H.D.P. and D.G.; Performed the experiments and analyzed the data, H.D.P., D.G., K.R., D.F., B.Z., P.Z., M.L. and K.G.M.; Contributed reagents/materials/analysis tools, K.G.M. and M.L.; Wrote the paper, H.D.P., D.G., D.F., M.L. and K.G.M. All authors have read and agreed to the published version of the manuscript.
Figure 1.
Spermatid individualization is disrupted in Z3-5009 mutant. Actin cone complexes in Z3-5009 testes (a) form in association with spermatid nuclei but do not progress past this stage (inset). In Oregon R control testes (b), actin cones form in association with sperm nuclei (inset 1), travel synchronously down the axonemes driving progress of spermatid individualization (inset 2, 3), and break down in a cystic bulge; phalloidin staining, red; DAPI staining, blue. (c) Classification of actin cone phenotype in control Oregon R (n = 13, black) and Z3-5009 mutant (n = 16, white); the average number of actin cones falling into each phenotypic class is reported. Bar 100 µm. * p < 0.05; *** p < 0.001.
Figure 1.
Spermatid individualization is disrupted in Z3-5009 mutant. Actin cone complexes in Z3-5009 testes (a) form in association with spermatid nuclei but do not progress past this stage (inset). In Oregon R control testes (b), actin cones form in association with sperm nuclei (inset 1), travel synchronously down the axonemes driving progress of spermatid individualization (inset 2, 3), and break down in a cystic bulge; phalloidin staining, red; DAPI staining, blue. (c) Classification of actin cone phenotype in control Oregon R (n = 13, black) and Z3-5009 mutant (n = 16, white); the average number of actin cones falling into each phenotypic class is reported. Bar 100 µm. * p < 0.05; *** p < 0.001.
Figure 2.
Individualization defects of Z3-5009 mutant visualized by electron microscopy. Cross-sectioned cysts of WT testis show individualized cysts (a); each of the control cysts contains pairs of axonemes and mitochondria derivatives enclosed by individual plasma membrane. Cross-sectioned cysts from Z3-5009 mutant show that individualization does not occur (c); numerous axonemes and mitochondria are enclosed within single membrane and surrounded by a large amount of cytoplasm, and the axonemes are not tightly juxtaposed to the mitochondria. Cross-sectioned cysts of flies that express the Pif1A-RI-GFP transgene and are homozygous for Z3-5009 (P[Pif1A-RI-GFP/CyO3; Z3-5009/Z3-5009]) show that the Z3-5009 phenotype is rescued—after individualization tightly juxtaposed axoneme/mitochondria pairs are surrounded by separate membranes (b). Bar 1 µm.
Figure 2.
Individualization defects of Z3-5009 mutant visualized by electron microscopy. Cross-sectioned cysts of WT testis show individualized cysts (a); each of the control cysts contains pairs of axonemes and mitochondria derivatives enclosed by individual plasma membrane. Cross-sectioned cysts from Z3-5009 mutant show that individualization does not occur (c); numerous axonemes and mitochondria are enclosed within single membrane and surrounded by a large amount of cytoplasm, and the axonemes are not tightly juxtaposed to the mitochondria. Cross-sectioned cysts of flies that express the Pif1A-RI-GFP transgene and are homozygous for Z3-5009 (P[Pif1A-RI-GFP/CyO3; Z3-5009/Z3-5009]) show that the Z3-5009 phenotype is rescued—after individualization tightly juxtaposed axoneme/mitochondria pairs are surrounded by separate membranes (b). Bar 1 µm.
Figure 3.
Immunocytochemical characterization of Z3-5009 mutant. Schematic representations of: whole Drosophila testis (a), newly forming actin cones in association with spermatid nuclei (b), early actin cones that have just moved a little away from the nuclei (c), myosin VI localization in newly forming/early control (WT Oregon R) cones (d), and quail localization in newly forming/early control cones (e); red box in (a) shows area-of-interest for all of the images in this figure, arrows in (a) indicate the direction of actin cones movement during spermatid individualization. Immunofluorescence comparative analysis of localization of selected proteins in control (f–i’) and Z3-5009 (j–m’) actin cones; phalloidin staining, green; antibody of interest, red; merge, yellow; DAPI staining, blue. Myosin VI localizes to the front of conical actin cones in Z3-5009 mutant ((j), arrows), consistent with the same-stage Oregon R cones ((f), arrows). Actin-binding quail appears to proceed towards the rear domains of the control (g) and Z3-5009 (k) cones as seen by red staining, leaving more intense actin (green) areas in the front of the cones (arrows in (g,k)). Spermatid axonemes are highlighted by poly-glycylated tubulin (h,l) and axoneme organization is normal in the Z3-5009 mutant (l) if compared to Oregon R (h). Alpha-tubulin appears to degrade to a similar extent in the same-stage Oregon R ((i’) vs. (i)) and Z3-5009 ((m’) vs. (m)) actin cones. Bar 5 µm.
Figure 3.
Immunocytochemical characterization of Z3-5009 mutant. Schematic representations of: whole Drosophila testis (a), newly forming actin cones in association with spermatid nuclei (b), early actin cones that have just moved a little away from the nuclei (c), myosin VI localization in newly forming/early control (WT Oregon R) cones (d), and quail localization in newly forming/early control cones (e); red box in (a) shows area-of-interest for all of the images in this figure, arrows in (a) indicate the direction of actin cones movement during spermatid individualization. Immunofluorescence comparative analysis of localization of selected proteins in control (f–i’) and Z3-5009 (j–m’) actin cones; phalloidin staining, green; antibody of interest, red; merge, yellow; DAPI staining, blue. Myosin VI localizes to the front of conical actin cones in Z3-5009 mutant ((j), arrows), consistent with the same-stage Oregon R cones ((f), arrows). Actin-binding quail appears to proceed towards the rear domains of the control (g) and Z3-5009 (k) cones as seen by red staining, leaving more intense actin (green) areas in the front of the cones (arrows in (g,k)). Spermatid axonemes are highlighted by poly-glycylated tubulin (h,l) and axoneme organization is normal in the Z3-5009 mutant (l) if compared to Oregon R (h). Alpha-tubulin appears to degrade to a similar extent in the same-stage Oregon R ((i’) vs. (i)) and Z3-5009 ((m’) vs. (m)) actin cones. Bar 5 µm.
Figure 4.
Activated caspase activity is normal in the Z3-5009 mutant. Activated caspases (green) are present throughout cysts in the Z3-5009 mutant (b), consistent with Oregon R (a). In control cysts, caspases accumulate in the cystic bulges ((a), white arrowhead) and waste bags ((a), yellow arrowhead). No cystic bulges ((b), white arrowhead) or waste bags ((b), yellow arrowhead) are seen in the Z3-5009 mutant, consistent with the observation that actin cones do not progress away from nuclei. Phalloidin staining, red; DAPI staining, blue. Bar 100 µm.
Figure 4.
Activated caspase activity is normal in the Z3-5009 mutant. Activated caspases (green) are present throughout cysts in the Z3-5009 mutant (b), consistent with Oregon R (a). In control cysts, caspases accumulate in the cystic bulges ((a), white arrowhead) and waste bags ((a), yellow arrowhead). No cystic bulges ((b), white arrowhead) or waste bags ((b), yellow arrowhead) are seen in the Z3-5009 mutant, consistent with the observation that actin cones do not progress away from nuclei. Phalloidin staining, red; DAPI staining, blue. Bar 100 µm.
Figure 5.
Ultrastructure of S1-decorated WT actin cones—longitudinal sections. Newly forming actin cones (in association with spermatid nuclei) are composed of actin bundles parallel to the longitudinal axis of the cone (a). A small actin meshwork is visible at the front of the early cones that have just moved a little away from the nuclei (b). Individualized actin cones have two structural domains (c,d): the rear domain is composed of actin bundles and the front domain is composed of a dense actin meshwork; as the cones move farther away from spermatid nuclei, the amount and extent of meshwork increases ((d) vs. (c)). Arrow in (a) indicates the direction of actin cones movement during individualization. Bar 1 µm.
Figure 5.
Ultrastructure of S1-decorated WT actin cones—longitudinal sections. Newly forming actin cones (in association with spermatid nuclei) are composed of actin bundles parallel to the longitudinal axis of the cone (a). A small actin meshwork is visible at the front of the early cones that have just moved a little away from the nuclei (b). Individualized actin cones have two structural domains (c,d): the rear domain is composed of actin bundles and the front domain is composed of a dense actin meshwork; as the cones move farther away from spermatid nuclei, the amount and extent of meshwork increases ((d) vs. (c)). Arrow in (a) indicates the direction of actin cones movement during individualization. Bar 1 µm.
Figure 6.
Longitudinal sections of S1-decorated Z3-5009 actin cones vs. cones from flies that express the Pif1A-RI-GFP transgene. Mutant actin cones (
a–
d) are composed of much shorter and thinner actin filaments than the WT cones (
Figure 6a–d vs.
Figure 5a–d). Newly forming Z3-5009 cones contained mainly parallel bundles of actin (
a). Some of the mutant cones, which appeared to move slightly away from the spermatid nuclei (
b), have disturbed structure with a bigger front meshwork and less organized rear region of parallel bundles than in WT early cones (
Figure 6b vs.
Figure 5b,c). Arrows in (
a,
b) indicate an abnormal actin meshwork that is denser on only one side of the early Z3-5009 cones. Most of the later mutant actin cones are disorganized (
c,
d). Longitudinal sections of actin cones from flies that express the Pif1A-RI-GFP transgene and are homozygous for Z3-5009 (p[Pif1A-RI-GFP/CyO3; Z3-5009/Z3-5009]) show that the Z3-5009 phenotype is rescued—the cones form and assemble actin properly during spermatid individualization (
e–
h). Arrow in h indicates the direction of actin cone movement during individualization. Bar 1 µm.
Figure 6.
Longitudinal sections of S1-decorated Z3-5009 actin cones vs. cones from flies that express the Pif1A-RI-GFP transgene. Mutant actin cones (
a–
d) are composed of much shorter and thinner actin filaments than the WT cones (
Figure 6a–d vs.
Figure 5a–d). Newly forming Z3-5009 cones contained mainly parallel bundles of actin (
a). Some of the mutant cones, which appeared to move slightly away from the spermatid nuclei (
b), have disturbed structure with a bigger front meshwork and less organized rear region of parallel bundles than in WT early cones (
Figure 6b vs.
Figure 5b,c). Arrows in (
a,
b) indicate an abnormal actin meshwork that is denser on only one side of the early Z3-5009 cones. Most of the later mutant actin cones are disorganized (
c,
d). Longitudinal sections of actin cones from flies that express the Pif1A-RI-GFP transgene and are homozygous for Z3-5009 (p[Pif1A-RI-GFP/CyO3; Z3-5009/Z3-5009]) show that the Z3-5009 phenotype is rescued—the cones form and assemble actin properly during spermatid individualization (
e–
h). Arrow in h indicates the direction of actin cone movement during individualization. Bar 1 µm.
Figure 7.
Identifying candidate genes for Z3-5009 phenotype. Cytological map of area-of-interest (chromosome 3R) derived by deficiency crosses. Top blue and purple rectangles refer to genetic regions that the deficiency lines are lacking—the sequences deleted extend beyond the constraints of this figure. Purple represents deficiency lines that failed to complement the mutant phenotype; blue represents lines that complemented (are fertile). In the lower portion, red arrows represent genes in the area-of-interest, and the orange arrow represents the gene thought to be responsible, Pif1A.
Figure 7.
Identifying candidate genes for Z3-5009 phenotype. Cytological map of area-of-interest (chromosome 3R) derived by deficiency crosses. Top blue and purple rectangles refer to genetic regions that the deficiency lines are lacking—the sequences deleted extend beyond the constraints of this figure. Purple represents deficiency lines that failed to complement the mutant phenotype; blue represents lines that complemented (are fertile). In the lower portion, red arrows represent genes in the area-of-interest, and the orange arrow represents the gene thought to be responsible, Pif1A.
Figure 8.
Pif1A overview and qPCR. Transcript map of the five predicted Pif1A transcripts (a). Exons of each transcript are shown in red; Pif1A-RI is the transcript used for rescue and targeting in RNAi. The SNP thought to be responsible for the phenotype is 45 bp downstream from the last codon of the Pif1A-RI mRNA transcript (b); it is a C>T transition. Relative Pif1A-RI mRNA expression levels in male testes (c); expression is decreased in Z3-5009 mutant. Error bars represent ± standard error.
Figure 8.
Pif1A overview and qPCR. Transcript map of the five predicted Pif1A transcripts (a). Exons of each transcript are shown in red; Pif1A-RI is the transcript used for rescue and targeting in RNAi. The SNP thought to be responsible for the phenotype is 45 bp downstream from the last codon of the Pif1A-RI mRNA transcript (b); it is a C>T transition. Relative Pif1A-RI mRNA expression levels in male testes (c); expression is decreased in Z3-5009 mutant. Error bars represent ± standard error.
Figure 9.
Spermatid individualization in bamGal4>Pif1A-RNAi testis and fertility assays. Actin cones are arrested with nuclear bundles in bamGal4>Pif1A-RNAi testis (a), consistent with the Z3-5009 mutant. Specifically, testes from these flies contained arrested actin cones associated with spermatid nuclei ((a), green arrowhead) or F-actin sleeves ((a), blue arrowhead) and no cystic bulges/waste bags are observed ((a), white arrowhead). Phalloidin staining, red; DAPI staining, blue. A small minority of the testes of this genotype appear roughly WT as explained in the text. Fertility assays (b)—all of the listed Drosophila lines represent males that were crossed with female virgin Oregon R flies; P[Pif1A-GFP], homozygote P[Pif1A-RI-GFP]. Numbers in parentheses refer to sample size. Error bars represent ± standard error. Bar 100 µm.
Figure 9.
Spermatid individualization in bamGal4>Pif1A-RNAi testis and fertility assays. Actin cones are arrested with nuclear bundles in bamGal4>Pif1A-RNAi testis (a), consistent with the Z3-5009 mutant. Specifically, testes from these flies contained arrested actin cones associated with spermatid nuclei ((a), green arrowhead) or F-actin sleeves ((a), blue arrowhead) and no cystic bulges/waste bags are observed ((a), white arrowhead). Phalloidin staining, red; DAPI staining, blue. A small minority of the testes of this genotype appear roughly WT as explained in the text. Fertility assays (b)—all of the listed Drosophila lines represent males that were crossed with female virgin Oregon R flies; P[Pif1A-GFP], homozygote P[Pif1A-RI-GFP]. Numbers in parentheses refer to sample size. Error bars represent ± standard error. Bar 100 µm.
Figure 10.
Pif1A localization time series. Schematic of localization of Pif1A protein throughout spermatid individualization (a); Pif1A (green) is present in the front region of nascent needle-shaped actin cones and is not observed in individualized cones. Representative homozygote P[Pif1A-RI-GFP] individualization complexes (b–g) stained with DAPI (blue), phalloidin/actin (red) and Pif1A-GFP (green) and representative MiMIC-GFP cones (h–m) stained the same way—localization profile is consistent with homozygote Pif1A-GFP transgenic cones. Bar 5 µm.
Figure 10.
Pif1A localization time series. Schematic of localization of Pif1A protein throughout spermatid individualization (a); Pif1A (green) is present in the front region of nascent needle-shaped actin cones and is not observed in individualized cones. Representative homozygote P[Pif1A-RI-GFP] individualization complexes (b–g) stained with DAPI (blue), phalloidin/actin (red) and Pif1A-GFP (green) and representative MiMIC-GFP cones (h–m) stained the same way—localization profile is consistent with homozygote Pif1A-GFP transgenic cones. Bar 5 µm.
Table 1.
Male fertility status of selected Drosophila deficiency lines.
Table 1.
Male fertility status of selected Drosophila deficiency lines.
Deficinecy Line | Fertility × OregonR |
---|
Df(3RBSC466 | Fertile |
Df(3R)BSC506 | Fertile |
Df(3R)BSC197 | Fertile |
Df(3R)Exe18143 | Sterile |
Df(3R)ED5330 | Sterile |
Df(3R)Exel6150 | Sterile |
Table 2.
Male fertility status of selected Drosophila RNAi lines.
Table 2.
Male fertility status of selected Drosophila RNAi lines.
RNAi Line | Gene of Interest | BamGal4>GOI-RNAi × OregonR |
---|
VDRC 51028 | CG33720 | Fertile |
P(KK110403)VIE-260B | CG13318 | Fertile |
W1118-, P(GD13849)v35860/TM3 | CG8223 | Fertile |
P(KK114195)VIE-260B | CG34135 | Fertile |
P(KK114379)VIE-260B | CG34301 | Fertile |
P(KK110554)VIE-260B | Pif1A | Sterile |
VCRD 51027 | Pif1B | Fertile |
P(KK112123)VIE-260B | CG11768 | Fertile |
P(KK107176)VIE-260B | CG8236 | Sterile |
P(KK106131)VIE-260B | CG33189 | Fertile |