PMC

Image showing the abnormally formed abdominal tergites (arrow) in an f07138 homozygous fly. Wings were deliberately held apart to show the abdomen clearly.

Western blot showing a comparable 74 kD band detected with the MAB1618 antibody in testes from wild type and the three Dic61B mutants (genotypes indicated above the lanes). The lower panel shows tubulin in the same blot as an internal control.

Clustal W alignment of the deduced amino acid sequence of Dic61B-PA (Dro) with axonemal dynein intermediate chain inner arm I1 of Culex (Cul) and Axonemal Dynein intermediate Chain 1 (DNAI1) of human (Hum). Note that Dic61B shares higher extent of homology towards the C terminal end with the other two proteins.

Confocal images showing various cell types of testes in wild type (A–D) and ms21 mutant (E–H) immunostained with anti Cdic antibody, MAB1618 (green) and DAPI stained chromatin (blue). Cytoplasmic distribution of Cdic was observed in hub cells (A, E), spermatocytes, (B, F), spermatids (C, G) and sperm bundles (D, H). The dot-like structures (arrows in A–C and E–G) might represent centrosomes, as Cdic is known to colocalize with centrosomes . Unindividualized sperm bundles showed strong Cdic expression (long arrows in D and H) whereas those with advanced individualization showed very weak staining (short arrow in D).

Chromatogram files of ms21 and wild type Dic61B gene sequences and the corresponding Blast analysis (ms21 is query, Q and WT is subject, S). Green highlighted base pairs in Blast analysis in panel A show the deletion of one Adenosine in ms21, in between positions +63 to +70. In panel B, the highlighted base pairs show base substitution (G→T) in ms21 at position +1203 in the fourth exon, which corresponds to +1204 in wild type (subject), as a single base is deleted in ms21, upstream to this position. These changes are marked by rectangles in chromatogram files above them.

Confocal images showing comparable organization of asters (green in A–B, detected with anti β tubulin, E7 antibody), centrosomes (green in C–D, detected with anti γ tubulin, GTU88 antibody), during meiosis I in spermatocytes and of the elongation cones at the caudal end of sperm tails (red in E–F, detected with anti α Spectrin 3A9 antibody), in wild type (A, C and E) and ms21 (B, D and F). Sperm tails are visualized with the help of α tubulin (DM1A antibody) staining in E–F (green). DNA is counterstained with DAPI (blue) in all panels.

Phase contrast images of intact (A–D) or lightly squashed (E–F) testes of wild type (A, E), ms21 (B, F), c05439 (C) or f07138 (D) male flies. Note the abnormally coiled sperm tails in mutants (thin arrows in B–D), while they are straight in wild type (arrow in A). Block arrows point to the misplaced cysts on the convex side of the testis in B and C and to the spermatid cyst preceding the spermatocyte cysts in D. The seminal vesicles (SV) are filled with sperms in wild type (A) but either filled with debris (B and C) or are empty (D) in the mutants. Motile and individualized sperms (block arrow) together with sperm bundles (arrow) are seen in partially squashed testes from wild type (E) but only unindividualized, immotile and bent sperm bundles are seen in testes from ms21 flies (arrow in F). Bar in A = 100 µm for A–D. Bar in E = 20 µm, for E–F.

Confocal images of Phalloidin (red) and DAPI (blue) stained testes from wild type (A, C, E) and ms21 mutant (B, D, F) flies showing the organization of individualization complexes (ICs). A–B, ICs assembled in the basal region of wild type and ms21 testes (short thin arrows in A and B, respectively). The ICs move synchronously towards the apical end where waste bags are formed in wild type testis but in ms21, the ICs manage to travel only till the middle region of testis (long arrows in A and B, respectively). Seminal vesicles in wild type are swollen due to storage of sperms but are very thin in ms21, being completely devoid of sperms (block arrows in A and B, respectively). C–D, Higher magnification images of ICs assembled normally in the basal region of wild type and ms21 testes respectively. E–F, Higher magnification images of waste bags formed at the apical end of testis on completion of individualization in wild type and the progressing ICs disrupted en-route to the apex in ms21 (arrows in E and F respectively). Bars in A, C and E represent 100, 20 and 10 µm, respectively.

Confocal micrographs of testes from DJ-GFP (A, C, E and G) and ms21 DJ-GFP (B, D, F and H) homozygous males showing sperm axonemes labeled with Don Juan-GFP protein (green), phalloidin stained ICs (red) and DAPI stained chromatin (blue). Sperm axonemes in wild type are straight (A, C, E and G, short arrow in A) but those in ms21 are bent and convoluted (B, D and H, short arrow in B). Long arrows in A and B indicate the seminal vesicles, filled with sperms in wild type and empty in ms21, respectively. Arrows in C and D show the normally assembled ICs around the sperm heads in the basal coil of wild type and ms21 testis, respectively. Arrows in E–F show progressed ICs separating the bundles of sperms into individual ones in wild type and ms21 respectively. Note that pearl like structures of DJ-GFP colocalize with the actin cones of ICs. Arrows in G and H show the waste bags formed near the apical region in wild type and disrupted ICs in ms21, respectively, since the ICs of ms21 get entangled in its abnormally coiled sperm tails. Bar in A is 100 µm (for A–B), bar in C is 20 µm for C–D and G–H. Bar in E is 10 µm, for E–F.

A, Schematic of an axial fiber of Drosophila sperm, adapted from Nielsen and Raff showing the typical 9+9+2 arrangement of microtubules (see text for details). B, Wild type post-individualization axonemes, each wrapped in its own membrane showing the typical 9+9+2 architecture. Each axial fiber in a sperm is associated with two mitochondrial derivatives, a major (M) and a minor (m) one (long and short arrows in B, respectively). C, ms21 axonemes from unindividualized sperm bundle showing various defects: in the axoneme on the left, both the mitochondrial derivatives are of the same size while in the axoneme towards right, m is larger than M, and the two are not properly orientated, unlike in wild type, where M is always larger than m and they are oriented in a particular angle with respect to the axial fiber. The radial spokes and the secondary fibers (sf, long white arrow) in both the axonemes of ms21 are not uniformly organized. Some of the secondary fibers in the axoneme towards right are enlarged and appear as tubules. Outer and inner dynein arms associated with some of the peripheral doublets in both axonemes are normal (short white arrows) but are reduced in some doublets (black arrows). Since none of the sperm bundle completes individualization in the ms21 testes, distinct cell membranes between different axonemes were not identifiable. Scale Bar in is 200 nm.

A. Schematic of Dic61B gene and primer pairs used for PCR analysis (primer pair numbers are in descending order in 5′ to 3′ direction of the gene). piggyBac 5′ and 3′ end specific primers were also used (not shown). B a–d, PCR amplicons obtained with primer pairs P1–P4, respectively, with genomic DNA of wild type and mutant lines (indicated on top of the PCR lanes). Note that all the three mutants generated amplicons similar to wild type with the P1 and P2 primer pairs (a–b), whereas the primer pairs 3 and 4 did not produce any amplicons with f07138 and c05439 genomic DNAs (c–d), respectively, due to the presence of piggyBac elements in these regions (see panel A). The ms21 genomic DNA produced amplicons similar to wild type with all the four primer sets (a–d). e–f, amplicons obtained with a combination of gene specific and pBac end specific primers with f07138 (PB5′-FP3) and c05439 (PB3′-FP4) genomic DNAs, respectively. g–h, amplicons of identical size obtained with genomic DNA of wild type and revertant flies (f07138^R and c05439^R) with primer pairs P3 and P4, respectively. pUC12 DNA digested with Hinf I was used as the marker in all cases.

A, Schematic of Dic61B-RA transcript and primer pairs used in RT-PCR analysis. The Dic61B gene has six exons (green) and five introns (grey), with their sizes shown below and above them, respectively. Primer pair, FP5-RP5 flanks the last (5^th) intron, FP5-RP6 pair flanks the 4^th and 5^th introns together, FP7-RP7 pair flanks the 2^nd and 3^rd introns together and FP7-RP8 pair flanks the first three introns of the gene. Only one isoform, Dic61B-RA is shown for simplicity. Dic61B-RB differs from RA in sizes of the first intron and second exon, which are of 42 bp and 176 bp, respectively (http://flybase.org). B (a–d), RT-PCR analysis of testes RNA from wild type and mutants (indicated on top of respective columns) with the different primer sets shown in panel A. a, Amplicons obtained with wild type genomic DNA (first lane) and wild type and mutant testis specific cDNA, with primer set FP5-RP5. Note that wild type cDNA generates an amplicon of smaller size as compared to its genomic DNA, due to removal of intron. The ms21 and c05439 cDNAs show unspliced amplicons. In f07138, the splicing is more or less comparable to wild type. b–d, Amplicons obtained with testis specific cDNA of wild type and mutants, with primer sets, FP5-RP6, FP7-RP7 and FP7-RP8, respectively. Note similar splicing defects in mutants with these primer sets also. No amplicon is seen in f07138 lane in panel c as the FP7-RP7 primer set flanks the piggyBac element in this allele (see panel A). The f07138 and c05439 alleles are not represented in panel d, since the primer pair FP7-RP8 flanks the transposons in both these alleles and hence no amplification is seen. e, 209 bp amplicon obtained with wild type genomic DNA and 141 bp amplicons obtained with wild type and mutant testis specific cDNAs with primers specific for GPDH internal control.