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First published online 9 February 2005
doi: 10.1242/dev.01672

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twin, a CCR4 homolog, regulates cyclin poly(A) tail length to permit Drosophila oogenesis

Jason Z. Morris^1,*, Amy Hong², Mary A. Lilly² and Ruth Lehmann^1,

¹ Developmental Genetics Program, Department of Cell Biology, The Skirball Institute and Howard Hughes Medical Institute, NYU School of Medicine, New York, NY 10016, USA
² Cell Biology and Metabolism Branch, National Institutes of Child Health and Human Development, National Institutes of Health, Bethesda, MD 20892, USA

View larger version (121K): [in a new window]   Fig. 1. twin egg chambers are defective in germ cell number and oocyte specification. Anterior is leftwards in all panels. (A-G) Anti-ORB antibody. (A'-G') DAPI. Arrowheads in A',C',E',F' indicate the karyosomes in the oocytes. (G'') Phalloidin. A twin mutant ovariole typically contains egg chambers exhibiting many of these phenotypes. (A,A') Wild-type chambers have 15 polyploid nurse cells and a posterior oocyte containing Orb and a condensed karyosome. (B,B') twinry5/Df. Many twin egg chambers die. These chambers do not stain with Orb antiserum and they have bright, punctate DAPI staining. Egg chamber death occurs in the germarium as well (not shown). (C,C') twinS3/Df egg chamber with five nurse cells and a normal oocyte. (D,D') twinS3/twinry5 egg chamber with two nurse cells and no oocyte. (E,E') A twinS3/twinry5 egg chamber with an otherwise normal oocyte in the center. (F,F') twinS3/Df egg chamber containing 14 nurse cells and two normal oocytes. (G,G',G'') twinry3/twinry5 egg chamber with 17 nurse cells and an oocyte in the center that has undergone five divisions. Five ring canals are present but one is not easily seen in this focal plane.

View larger version (50K): [in a new window]   Fig. 2. Mapping, cloning and expression of twin. (A) Genomic region around the ry3 and ry5 P-element insertions. The insertions (black arrow) fall in a 15 kb intron of ccr4. Distal probe: 1.5 kb of genomic DNA isolated from inverse PCR generated from the 5' end of the ry3 or the 3' end of the ry5 strains. Proximal probe: 1.5 kb of genomic DNA isolated from inverse PCR generated from the 3' end of the ry3 or the 5' end of the ry5 strains. The three exons of ccr4 included in the distal probe are shown. The proximal probe lies in the 15 kb intron. (B) Different splice forms of Drosophila ccr4 represented by ESTs isolated by the Drosophila Genome Project. The three exons preceding the 15 kb intron containing the P insertions are shown in gray. M, putative start methionine; (M), start methionine less certain because there is no in-frame stop codon upstream in the EST isolated; *, stop codon. (C) Northern blot of poly(A)-selected RNA from wild-type and twinry3/twinry3 ovaries. The distal and proximal probes are as described in A. RP49 was used as a loading control. The proximal probe seems to be upregulated in twinry3 homozygotes, but this is unlikely to contribute to the twin phenotype. The alleles are all recessive and phenotypically similar, and the point mutants have missense mutations in ccr4 and not in the predicted ORFs of the intron. (D-K) mRNA in situ hybridization. Anterior is leftwards in all panels. (D-G,I-J) ccr4 antisense probe. (H,K) ccr4 sense negative control. (D) Maternal ccr4 is present at very high levels in early embryos. (E) Expression is quite reduced in the blastoderm. (F,G) Expression is general, with strong staining in the central nervous system. (H) No staining observed in sense control. (I,J) Expression is present throughout the ovary, with stronger expression in germ cells than in soma. Expression is particularly strong in late (stage 10 and later) nurse cell. Staining in germarium region 1 was not strong, but this tissue is refractory to in situ hybridization. (K) No staining observed in sense control.

View larger version (33K): [in a new window]   Fig. 3. The S1 and S3 alleles disrupt highly conserved amino acids in Drosophila Ccr4. (A) Cartoon showing the domains present in DNOCTURNIN, ANGEL and TWIN/CCR4, as well as S. cerevisiae CCR4. The LRR domains are not present in ANGEL and DNOCTURNIN. Five amino acids, corresponding in Drosophila CCR4 to N199, E243, D412, D486 and H526 have been shown to be essential for catalysis in S. cerevisiae (numbered 1-5) (Chen et al., 2002). (B,C) Black boxes: amino acids identical in all three species. Gray boxes: amino acids identical in two of the three species. Numbers designate the position of amino acids in their primary sequences. (B) Alignment of the leucine-rich repeat regions (LRRs) of CCR4 from Drosophila, humans and S. cerevesiae. S. cerevisiae has five LRRs, humans and Drosophila have half of the first LRR and the second, third and fourth LRRs. Deletions of yeast LRRs 4 and 5 have no effect (Clark et al., 2004). The invariant asparagine at the end of repeat 3 is translated as an isoleucine in the twinS1 allele. (C) Alignment of a region of the sequence between catalytic amino acids 2 and 3 from A, including the site of the S3 lesion. An isoleucine conserved in humans and Drosophila is translated as a serine in the twinS3 allele. S. pombe also has an isoleucine at this position (not shown), although S. cerevisiae has a valine.

View larger version (88K): [in a new window]   Fig. 4. CycA and CycB are misregulated in twin. Anterior is leftwards in all panels. The pair in each row was stained and photographed in parallel. (A-D) CycA, red; fusome (1B1), green. (E,F) CycB, red; fusome, green. (A) Wild-type (twin/+) chamber showing a cyst in region 1 expressing CycA. Some of the CycA co-localized with the fusome, which appears yellow. Posterior to this cyst are several highly branched fusomes. (B) twinS1/Df germarium showing very strong CycA staining throughout. CycA colocalizes with some fusomes even towards the posterior of the germarium (yellow). Fusomes are present, and contain branches. (C) Wild-type (twin/+) germarium showing CycA in two cysts in region 1. One of these cysts colocalizes CycA to the fusome. (D) twinry3/Df germarium showing CycA staining throughout. The misexpression phenotype is not as prominent as in the strongest allele, S1. The fusomes are clearly visible and highly branched. (E) Wild-type (twin/+) germarium showing CycB in two cysts in region 1. Both of these cysts show CycB co-localizing with the fusome. (F) twinS1/Df showing very strong CycB staining throughout germarium. The unstained regions are sites of dying cysts. CycB colocalizes with the fusome in all the cysts. Staining with anti-CycE antisera did not reveal a difference between twin/Df and twin/+ germaria (not shown).

View larger version (79K): [in a new window]   Fig. 5. Cytoplasmic Bam is reduced in twin germaria. (A,B) BamC, red; germ cells (Vasa), green. (A',B') red channel alone. The BamC antiserum is specific to the cytoplasmic form of Bam (McKearin and Ohlstein, 1995). Bam can also localize to the fusome, but that fraction is not detected by the available antibodies. Vasa is expressed in germ cells. Anterior is leftwards in all panels. All samples were stained and photographed in parallel. (A,A') Wild-type (OR) germarium showing BamC staining in a cyst in Region 1. All the germ cells express Vasa. (B,B') twinS1/Df germarium showing very reduced BamC staining. twin heterozygotes exhibit BamC staining in dividing cysts, but also exhibit high background staining (not shown).

View larger version (64K): [in a new window]   Fig. 6. cycA, cycB and cycE messages have longer poly(A) tails in twin mutants. In all panels, + and - refer to the presence or absence of reverse transcriptase (RT) in the reaction. Left brackets show sizes of most abundant poly(A) tails in twinS1/Df ovaries. Right brackets show sizes of most abundant poly(A) tails in twin/+ ovaries. (A) cycA mRNA. Two different amounts of wild-type (twin/+) RNA are loaded to show the dramatically different distributions of poly(A) tail length in twin mutant versus wild type. The predominant class of poly(A) tail lengths in twinS1/Df is from 40-80 bp long. The predominant class of poly(A) tail lengths in twin/+ is from 0-20 bp long. (B) The distribution of lengths of cycB poly(A) tails is similar in twinS1/Df and twin/+. The poly(A) tails may be slightly higher in the twin homozygotes. (C) cycE poly(A) tails tend to be significantly longer in twinS1/Df than in twin/+. (D) The distribution of bam poly(A) tail length is similar in twin mutant versus wild type. Short tails are more abundant in both genotypes and poly(A) tails reach the same maximum length, although there may be more of the very shortest tails in the wild type. A slow-migrating band is more abundant in twinS1/Df than in twin/+, but as the band is isolated far above the distribution of poly(A) tails and as BamC levels are lower in twinS1/Df, this is probably not a reflection of deadenylation defects.