Figure 1.
pex31 and pex161 lack normal peroxisomes.
(A) Genomic lesions induced in pex3 and pex16. Untranslated regions and coding regions are shown as open and filled boxes, respectively. The P-element insertion sites in GS9829 and GS14106 are indicated by triangles. Genomic regions deleted in the mutants are indicated by dashed lines. Scale bar represents 0.5 Kb. (B–G) Peroxisomes detected in vivo. Malpighian tubules dissected from the larvae of wild-type (B, E), pex31 homozygotes (C, F), and pex161 homozygotes (D, G), ubiquitously expressing UAS-EGFP-SKL (B–D) or UAS-PMP70-ECFP (E–G) driven by Act-GAL4 are shown. Insets at upper right are higher-magnification images of the area enclosed by broken-line squares. Scale bars in B and E represent 10 µm. (H and I) Electron micrographs of adult malpighian tubule sections stained with DAB in wild-type (H) and pex161 homozygous flies (I). Arrowheads in H indicate DAB-stained peroxisomes. Scale bar in H represents 1 µm.
Figure 2.
The pex16 mutant showed a reduced body size, rosy eyes, and the accumulation of VLCFAs.
(A and B) The pex161 homozygote was smaller than wild-type. An adult female (A) and male (B) pex161 homozygote (−/−) and heterozygote (−/+) are shown. (C) Mean body weight of individual adult flies. Genotype: (−/Df), pex161/Df(3L)ri-XT1; (−/+), pex161/TM3; (Df/+), Df(3L)ri-XT1/Dr; (+/+), Dr/TM3. Error bars represent standard deviations. (D–F) pex16 homozygotes had rosy eyes. Eyes of wild-type (D), a pex161 homozygote (E), and a rosy506 homozygote (F) are shown. (G) VLCFAs accumulated in the pex161 homozygote. The fatty acid content of adult males homozygous for pex16 was measured by gas chromatography three days after eclosion. Each bar represents the average level of each VLCFA component in pex161 homozygotes compared to that in wild-type flies, obtained from three independent experiments. Error bars represent standard deviations.
Figure 3.
pex16 homozygotes showed defects in neural functions and development reminiscent of PBD-associated symptoms.
(A and B) Climbing assay scores with increasing age (days after eclosion). (A) Genotypes: (+/+), wild-type; (−/+), pex161/+; (−/−), pex161/pex161; (−/Df), pex161/ Df(3L)ri-XT1. Averages of three trials are shown. (B) Climbing scores with the wild-type pex16 gene driven by the indicated GAL4 drivers (key shown at right) in pex161 homozygotes. GAL4 drivers: elav, elav-GAL4 (expressed in differentiated neurons); Lsp2, Lsp2-GAL4 (expressed in the fat body); NP5021, a GAL4 line expressing GAL4 ubiquitously in the gut. (C and D) Flight activity scores. Fight levels (1–7) plotted against the percentage of flies at each level are shown. Each trial included 20 flies. (C) Genotypes: (−/+), pex161/+; (−/−), pex161/pex161. Flies were examined two (2 days) and ten (10 days) days after eclosion. (D) Flight scores when the wild-type pex16 gene was driven by the indicated GAL4 drivers in pex161 homozygotes (see description of drivers in B). Flight activities were measured two days after eclosion. (E and F) Longevity of adult male flies. Percentage of survivors (n = 20) at 25°C was determined every three days. (E) Genotypes were as described in A. (F) Longevity when the wild-type pex16 gene was driven by the indicated GAL4 drivers (described in B) in pex161 homozygotes. (G–L) pex161 homozygotes showed defective neural development. Brains from adult male flies one day after eclosion were stained with the 22C10 antibody to reveal the axonal and dendritic structures. Confocal images of the optic lobe are shown: wild-type (G), pex161 homozygote (H), pex161 homozygote overexpressing wild-type pex16 driven by elav-GAL4 (I), Lsp2-GAL4 (J), elav-GAL4 and Lsp2-GAL4 (K), or NP5021 (L). Insets are higher magnifications of the areas enclosed by broken lines. Arrowheads indicate spongy structures.
Figure 4.
The maturation of germline cells is arrested in the testis of pex16 homozygotes.
(A) Schematic representation of early spermatogenesis. Germline stem cells (s) and somatic cyst progenitor cells, anchored to somatic hub cells, produce a new stem cell and a gonialblast (g), which are enclosed by two somatic cyst cells (Cyst). A single gonialblast cell undergoes four rounds of mitotic divisions to produce 16 early spermatocytes that then enter the growth phase. After the growth phase, the late spermatocytes undergo two meiotic divisions, producing 64 haploid spermatids. (B–E) Phase-contrast micrographs of the wild-type (B and C) and pex161 homozygote (D and E) testes. The bundles of elongated spermatids (arrow in B) and postmeiotic spermatids (inset in C) were missing in the pex161 homozygote testis. Inset in C is a higher magnification of the area enclosed by broken lines. Scale bars represent 100 µm.
Figure 5.
Maturation of spermatocytes is arrested in the spermatocyte growth phase.
(A) Schematic representation of spermatocyte growth phase. Nu, nucleolus; MT, mitochondria. (B–G) Maturation of spermatocytes in the testes of wild-type (B–E) and pex161 homozygote (F–G) flies. Y-chromosome loops and chromatin were stained with the S5 antibody (center panels) and Hoechst 33258 (right panels), respectively. Polar spermatocytes (B and F) and young apolar spermatocytes (C and G) were observed in both wild-type and pex161 mutant testes, whereas mature spermatocytes (D) and spermatocytes with meiotic chromosome condensation (E) were observed only in wild-type testes. Degenerated spermatocytes (arrows in H) were observed in the pex161 homozygote testes in squash preparations of live cells. Scale bar represents 20 µm.
Figure 6.
Peroxisomes in somatic cyst cells are essential for male germ cell maturation.
(A–D) Phase-contrast micrographs of a pex161 homozygote testis expressing UAS-pex16 and UAS-EGFP-SKL driven by ptc-GAL4, expressed in cyst cells (A and B) or nos-GAL4, expressed in germline cells (C and D). Elongated spermatids (arrow in A) and morphologically normal spermatids (arrow in B) are indicated. (B′ and D′) Peroxisomes detected by EGFP-SKL. The fluorescent images in B′ and D′ correspond to the phase-contrast micrographs of B and D, respectively. Insets showing single spermatocytes are higher magnifications of the areas enclosed by broken lines. Scale bars represent 100 µm.