Essential and recurrent roles for hairpin RNAs in silencing de novo sex chromosome conflict in Drosophila simulans
Fig 3
Cytological basis of male reproductive defects in nmy and tmy knockouts.
(A, B) Confocal images of whole mount testes from control flies (w[XD1] as wild-type control) and mutants (nmy, tmy, and ago2) stained with a pan-Histone antibody (red) and phalloidin (F-actin green) to reveal ICs and DAPI to label nuclei (blue). (A) Whole testis images, with the stem cell regions labeled with asterisks and seminal vesicles labeled with SV. (A) Control w[XD1] testis shows the orderly progression from mitotic (1), meiotic (2), and differentiation (3) regions. (B) nmy mutants exhibit grossly normal spermatogenesis with normal ICs (arrows, A’). Spermatogenesis is highly disturbed in tmy and ago2 homozygous mutants, with disorganized cysts and aberrant (tmy) or absent (ago2) IC (A’). The empty SVs. Scale bars: 100 μm. (B) Focus on the SV shows they are filled with sperm in control and nmy mutants, as seen with needle-shaped DAPI staining, but are empty in tmy and ago2 mutants; the large round nuclei at the borders correspond to the cells of the somatic wall. (C, D) Cysts of 64 spermatids in control (w[XD1]) and nmy mutants (grown at 18°C). The stages of the histone-to-protamine transition indicated were determined with pan-Histone staining and nuclear shape. Histones are eliminated in both control (C) and nmy (D) spermatids. However, individualization of control spermatid nuclei yields regular needle shapes that stain uniformly with DAPI, whereas about half of nmy mutant nuclei fail to elongate and exhibit abnormal shape (arrow). In addition, DAPI staining in nmy mutant nuclei is uneven, with both less (triangle) or more (DAPI foci, arrowhead) dense regions, suggesting aberrant chromatin organization. Scale bars: 10 μm. (E, F) Meiosis in control and tmy homozygous testes stained for histones (magenta) and α-tubulin (green). The stages indicated were determined by counting nuclei number in cysts (16 for meiosis I and 32 in meiosis II). In tmy mutants, chromosomes are fragmented and chromatin bridges are observed in anaphase and telophase of both meiosis I and meiosis II. Scale bars: 10 μm. IC, individualization complex; SV, seminal vesicle.