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doi: 10.1242/10.1242/dev.00513

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Drosophila TGIF is essential for developmentally regulated transcription in spermatogenesis

¹ Department of Anatomy, University of Cambridge, Downing Street, Cambridge, CB2 3DY, UK
² Department of Zoology, Oxford University, South Parks Rd, Oxford, OX1 3PS, UK

View larger version (85K): [in a new window]   Fig. 1. TGIF is represented in Drosophila by Achi and Vis. (A) Sequence comparison shows the high TGIF similarity within the homeodomain and extending into the C-domain. The TALE amino acids, RYN, characteristic of the TGIF family of TALE homeodomains, are boxed. The extended helix 3 is shown as predicted by PSIpred. The vertical line indicates the COOH-terminal limit of the homeodomain; * indicates identical residues or highly conserved substitutions in all sequences;. indicates less conserved sequences and! indicates conservation only between Achi/Vis and TGIF2. (B) Achi and Vis are highly similar and comparison with the Anopheles TGIF homologue (AgCP3385) indicates conserved regions in addition to the homeodomain and C domain. * indicates identity;: indicates highly conserved and. indicates conserved residues. The extents of the homeodomain and C domain are indicated above the sequence. The position of the insertion of exon 6 sequence is indicated by arrowhead. Arrows: number 1 indicates start of deletion in visZ3922 and number 2 indicates site of stop codon in achiZ3922. (C) Exon 6 sequence from class 2 transcripts. Exon 6 sequence is identical in achi and vis.

View larger version (41K): [in a new window]   Fig. 2. Gene structure and expression. (A) achi and vis are tandem duplications. The intron/exon structure is shown together with the alternative splice products determined from completely sequencing the following cDNAs: achi-class 1 LD25085,GM01582; achi-class 2 LP02076; vis-class 1 SD08875, SD01238. vis-class 2 intron/exon structure was determined by sequencing a subclone of the ORF RT-PCR shown in D. Translation is initiated in exon3 (arrow) and terminates in exon10 (asterisk). (B) RT-PCR analysis with primers (f1 and r3) common to both achi and vis indicates expression throughout development. A similar result was obtained with primers specific for achi. The RP49 control gave approximately equal levels with RNAs from all the different developmental time points. (C) Sex-specific splice differences of achi/vis. Class 2 products predominate in testes (lane 1) whilst class 1 products (lane 2) predominate in ovaries (primers: f3 and r2). Similar results are seen comparing male and female whole fly RNA (not shown). (D) Class 2 transcripts are testes specific. Lane 1: testes; lane 2: male gonadectomised carcass and lane 3: adult females (achi/vis ORF primers).

View larger version (17K): [in a new window]   Fig. 3. Deletion mutants. Position of the starting transposon insertion (EP(2)2107) is indicated. EP(2)2107 homozygotes are viable and fertile although expression of achi class 1 transcripts is reduced. The Df(2R)achi1 deletion extends at least to the 3' end of vis exon 4. Prediction of genes in the region is based on release 3 of the genome annotation. Genes transcribed left to right in the figure are above the line, those in the right to left orientation are below the line.

View larger version (62K): [in a new window]   Fig. 4. Loss of achi/vis function results in male infertility. (A) Schematic of spermatogenesis; (a) stem cell at apex of testis, (b) zone of mitotic expansion of spermatogonia, (c) primary spermatocytes and meiotic divisions, (d) differentiated sperm. (B-D) Phase contrast image of (B) wild type (arrow indicates bundles of differentiated sperm); (C) Df(2R)achi1 homozygous testis (note absence of sperm and abundance of undifferentiated primary spermatocytes; arrow); (D) achiZ3922 visZ3922; same phenotype as Df(2R)achi1; (E) In situ hybridisation with probe recognising the products of both achi and vis. Expression is low/undetectable at the apex (arrow), high in the primary spermatocyte stage and persists into the meiotic divisions.

View larger version (75K): [in a new window]   Fig. 5. The meiotic arrest phenotype. A. DAPI labelling is strong in the cells at the apex (arrow) and in Df(2R)achi1 homozygotes this zone is expanded (B). (C-F) Anti-histone labelling. In the wild type (C) and in mia homozygotes (F) there is a small population of early primary spermatocytes (arrow in C) and this population is markedly expanded in Df(2R)achi1 (D; arrow) and aly homozygotes (E).

View larger version (113K): [in a new window]   Fig. 6. achi/vis phenotype. (A-D) The late stage primary spermatocyte chromatin phenotype. (A) Phase contrast image of wild-type stage 5 primary spermatocyte. Arrow indicates nucleolus, arrowhead indicates Y-chromosome loops. (B) DAPI labelling reveals the chromatin peripherally located against the nuclear membrane. (C) Primary spermatocyte from Df(2R)achi1 homozygote testis demonstrating stage 5 characteristics: nucleolus (arrow), Y-loops (arrowhead) and intact nuclear membrane. (D) DAPI labelling of cell in C reveals chromatin clumps reminiscent of meiotic division stages (Cenci et al., 1994). (E-G) Anti-histone labelling reveals the chromatin configuration in stage 5 primary spermatocytes in wild type (E), Df(2R)achi1 (F) and aly (G). The chromatin morphologies in F and G are clearly distinct; the aly configuration resembles that of wild type, but is more diffuse. (H-K) Expression and localisation of Aly and Comr in achiZ3922 visZ3922 homozygote testes. (H) Wild type, anti-Aly; (I) achiZ3922 visZ3922, anti-Aly, (J) wild type, anti-Comr, (K) achiZ3922 visZ3922, anti-Comr. Aly and Comr are present in the nucleus in achiZ3922 visZ3922 testes but the localisation appears more diffuse than in the wild type.

View larger version (24K): [in a new window]   Fig. 7. Gene expression profile in Df(2R)achi1 mutants. RT-PCR analysis on RNA samples from wild-type and Df(2R)achi1 mutant testes.

View larger version (62K): [in a new window]   Fig. 8. Expression of esc in Df(2R)achi1 mutants. (A) RT-PCR analysis on RNA samples from wild-type and Df(2R)achi1 mutant testes reveal that esc is amongst the genes showing enhanced transcript abundance in Df(2R)achi1 mutants, other enhanced transcripts include haywire and Taf24. (B) Anti-Esc immunolabelling on wild-type testes. Esc accumulates in nuclear speckles during primary spermatocyte differentiation. (C) Anti-Esc immunolabelling on Df(2R)achi1 testis. Esc is clearly expressed and demonstrates the expansion of the early primary spermatocyte population. Nuclear speckles do not form.

View larger version (140K): [in a new window]   Fig. 9. Expression of TGIF in mouse testes. (A) Toluidine Blue stained cross section of adult mouse seminiferous tubules. Arrow, spermatogonia; arrowhead, dividing primary spermatocytes; asterisk, elongating spermatids. (B) Immunolabelling with anti-TGIF. Arrow indicates nuclear labelling in peripheral cells (spermatogonia).