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doi: 10.1242/10.1242/dev.00246

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Transcriptional activation in Drosophila spermatogenesis involves the mutually dependent function of aly and a novel meiotic arrest gene cookie monster

Department of Zoology, Oxford University, South Parks Rd, Oxford, OX1 3PS, UK

View larger version (152K): [in a new window]   Fig. 1. comrz1340 testes show a meiotic arrest phenotype. (A,B) Phase contrast micrographs of wild-type (A) and comrz1340 (B) mutant testes. Wild-type testis contains stages of spermatogenesis from mitotic spermatogonia (small arrow), primary spermatocytes (in region indicated by black line adjacent to testis), meiosis (large arrow) and spermatid elongation (arrowheads). comrz1340 testes contain mitotic spermatogonia (small arrow) and primary spermatocytes (line). Later stages are absent; instead, the basal region of the testis contains refractile necrotic cells (arrowhead). (C,D) Chromosomes of live wild-type (C) and comrZ1340 (D) primary spermatocytes visualised by staining with vital Hoechst. The arrows indicate the three major chromosome bivalents within one nucleus. The mutant chromosomes look fuzzy compared with wild type.

View larger version (75K): [in a new window]   Fig. 2. comr is more like aly than the can-class meiotic arrest genes. RNA in situ hybridisation of wild-type (A,E,I), aly5 (B,F,J), mia (C,G,K) or comrz1340 (D,H,L) testes, using probes against polo, boule and Mst87F, as indicated. Whole testes are shown, with the apical regions to the right-hand side of each panel; dark grey staining indicates presence of the transcript. Wild-type testes expressed polo in primary spermatocytes (A); polo mRNA was present in all the mutant testes (B-D). boule mRNA was expressed in wild-type primary spermatocytes, and the transcript persisted into early elongation stages (E). boule transcript was visible in mia primary spermatocytes (G), but was not detected in aly5 or comrz1340 mutant testes (F,H). Mst87F mRNA accumulated in wild-type primary spermatocytes and persisted until late stages of elongation (I). No accumulation of Mst87F transcript could be detected in any of the mutant testes (J-L).

View larger version (12K): [in a new window]   Fig. 3. comr genomic region showing overlapping deficiencies and predicted genes. The genomic region 57F-58A is shown in the centre of the figure. Deficiency chromosomes are shown above the region. Grey indicates deleted segments; black indicates non-deleted chromosomal regions; hatched boxes represent uncertainty. The predicted genes from this region are shown underneath: black boxes indicate the extent of the predicted gene; arrows indicate orientation. comr mapped to the overlap of Df(2R)EGFR3 and Df(2R)X58-7, between CG9284 and PpN58A. A small deletion was detected in Df(2R)XE-2900, which uncovers CG13493. Df(2R)XE-2900 also deletes a larger genomic region just proximal to that shown in this figure (indicated by the hatched box).

View larger version (51K): [in a new window]   Fig. 4. Comr predicted protein sequence. (A) Conceptual translation of Comr protein. The mutated amino acid in comrz1340 Q12* is indicated in an oval. The very acidic region at the C terminus of the protein is double underlined. Putative nuclear localisation signal is single underlined. The potential PB1-like domain is indicated in italics. Peptide used for antibody production is indicated in bold. (B) ClustalW alignment of the PB1 domain from human KPCI with the PB1-like region from Comr. Identical residues are indicated with vertical lines, very conservative substitutions with double dots, conservative substitutions with single dots.

View larger version (63K): [in a new window]   Fig. 5. comr is expressed most highly in early primary spermatocytes. (A) RT-PCR of comr from wild-type whole males (M), testis (T), a male carcass lacking testis (C) and whole females (F). In males, the transcript was testis specific. Very low levels of transcript were detected in the adult female sample. (B-D) RNA in situ hybridisation using a comr probe revealed the transcript was most abundant in early primary spermatocytes in wild type (B). Transcription of comr was unaffected in aly5 (C) or can3 (D) mutant testes.

View larger version (54K): [in a new window]   Fig. 6. Comr protein is nuclear in primary spermatocytes. (A) Western blot with the anti-Comr peptide antibody against testis extracts from wild-type (WT), aly5, comrz1340 and mia testes. The anti-Comr antibody recognised two bands, one at just under 100 kDa, the other at 120 kDa. The 120 kDa antigen was detected in all the samples, whereas the 100 kDa antigen (indicated by an asterisk) was absent from comrz1340 mutant testes. This corresponds to Comr protein. (B,C) Immunohistochemistry using the anti-Comr antibody revealed a spotty localisation in the wild-type testes, which corresponds to nuclear staining of the primary spermatocytes. Only background nonspecific staining of comrz1340 could be detected under these conditions. (D-I) Fluorescence staining of anti-Comr (E,H; green in merged images) and DNA (D,G; red in merged images). In wild-type primary spermatocytes, Comr protein was found throughout the nucleus, but was concentrated in several distinct domains (E). The arrowhead indicates specific Comr staining, overlapping the regions of highest DNA staining intensity. The arrow indicates bright staining attributed to the crossreacting band, not associated with strong DNA staining. comrz1340 mutant spermatocytes had much lower intensity staining in the chromosomal regions with the anti-Comr antibody (H, arrowhead). Residual staining was restricted to a small region in each nucleus not associated with DNA (H, arrow).

View larger version (104K): [in a new window]   Fig. 7. The nuclear localisations of Aly and Comr are mutually dependent. (A,B) Immunohistochemistry using an anti-Aly antibody. In wild-type (A) testes, Aly protein localised to the nuclei of maturing primary spermatocytes (arrow). In comrz1340 mutant testes (B) the staining of primary spermatocytes was exclusively cytoplasmic (arrow). (C-H) Fluorescence staining with anti-Comr (D,G; green in merge) and DNA (C,F; red in merge). In aly5 mutant spermatocytes (C-E) Comr protein was found only in one spot in each nucleus. In mia mutant spermatocytes, Comr protein was found throughout the nucleus (F-H).

View larger version (60K): [in a new window]   Fig. 8. mRNA transcription is associated with a subset of the Comr-positive chromatin. Triple labelling of DNA (A; red in merged images, D,E,G), Comr (B; green in merged images, D,F,G) and P-CTD (C; blue in merged images, E-G) in part of a cyst of wild-type primary spermatocytes. The nonspecific staining of the anti-Comr antibody is indicated by arrows in B. (D) Most but not all Comr staining associates with DNA. Arrowhead in D indicates a region of anti-Comr staining that does not colocalise with strong DNA staining. The arrow in D indicates a region of colocalisation of Comr with DNA. (E) Strong P-CTD reactivity was not associated with strong DNA staining. The arrowhead in E denotes a chromatin domain that is adjacent to, but not co-localised with, P-CTD reactivity. The region indicated by the arrowhead shows a region of DNA staining accompanied by weak P-CTD staining. (F) Comr staining was present in regions of mRNA transcription. Some regions of high transcription also had high Comr staining (arrows), other transcriptionally active regions had weaker Comr staining (arrowheads).

View larger version (24K): [in a new window]   Fig. 9. Model for meiotic arrest gene product action at target promoters. Target promoters may be dependent solely on the function of the aly-class genes, including aly and comr (e.g. twine), or they could additionally depend on the can-class genes (e.g. Mst87F). The promoter regions of these genes differ, here denoted by shaded or hatched enhancer elements (E) 5' of the TATA box. The aly-class proteins Aly and Comr would work together to promote local chromatin remodelling (small boxes on DNA), which would allow binding of specific transcription factors [TF(a) or TF(b)] to the enhancer elements. Binding of these transcription factors allows transcriptional activation by recruitment of the ubiquitous (u) TFIID in the case of can-independent genes, or testis specific (t) TFIID, including Can, in the case of can-class dependent genes.