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First published online 4 July 2007
doi: 10.1242/dev.003517

Development 134, 2851-2861 (2007)
Published by The Company of Biologists 2007
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A novel eIF4G homolog, Off-schedule, couples translational control to meiosis and differentiation in Drosophila spermatocytes

Tina M. Franklin-Dumont1, Chandrima Chatterjee1, Steven A. Wasserman2 and Stephen DiNardo1,*

1 Department of Cell and Developmental Biology, University of Pennsylvania School of Medicine, Philadelphia, PA 19104-6048, USA.
2 Section of Cell and Developmental Biology, University of California, San Diego, La Jolla 92093, USA.


Figure 1
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  		Fig. 1. ofs mutants fail to undergo meiosis or differentiation. (A-C) Phase images, apical-tip oriented to the left. (A) Heterozygous testes appear wild-type, with elongating spermatids filling most of the testis. (B) Mutant testes lack elongating spermatids. (C) ofs/Df(3R)mbc-R1 is indistinguishable from ofs/ofs (B). (D,E) High-magnification, with diagrams of the represented stages. Color code: primary spermatocyte nucleus, red; nucleolus, black; haploid spermatid nucleus, orange; fused mitochondrial derivative, purple; dissociated mitochondria, blue; cell outlines (where discernable), gray. (D) Heterozygous testes have examples of mature primary spermatocytes (left) and onion-stage spermatids (right). (E) The most prevalent cells found in ofs testes resemble primary spermatocytes. (F,G) Whole-mount immunofluorescent images of wild-type (F, arrowhead, 2xlacZ+) and ofs (G, arrow, lacZ-) clones in a heterozygous background. Flies were aged 4 days after clone induction, which allowed clones to progress midway through the meiotic G2 of wild-type spermatocytes. (F',G') Brackets are the same size in each panel, highlighting the cell size difference between the two clones. The entire ofs cell outline (PTyr, phospho-Tyrosine) is approximately the same size as the nucleus of the control cell.

 

Figure 2
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  		Fig. 2. ofs identifies a distinct pathway controlling spermatocyte development. (A,B) Immunofluorescent images of frozen split squashes. Aly staining (red) is indistinguishable between heterozygotes (A) and homozygotes (B). (A) Cyclin A (green) is predominantly cytoplasmic in heterozygous spermatocytes. (B) In the mutants, Cyclin A accumulates in nuclei (arrows) relatively close to the apical tip.

 

Figure 3
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  		Fig. 3. ofs mutants exhibit altered timing of Cyclin A, Boule and Twine accumulation. (A-D) Whole-mount immunofluorescence. (A,B) ofs clones (GFP+, green) in a heterozygous background. (A) ofs clones precociously exhibit nuclear Cyclin A (CycA, arrows, red), whereas heterozygous spermatocytes are only beginning to accumulate Cyclin A, and nuclear entry occurs only in cysts entering the G2-M transition (arrowhead). (Inset i) In early spermatocytes, the degree of chromatin condensation in ofs clones (outlined, upper right) is similar to heterozygous cells (left). (Inset ii) By contrast, older mutant cysts retain less condensed chromatin (outlined, upper right), whereas heterozygotes begin to condense chromatin as they approach meiosis (left-most and lower cells). (B) Midsection of the testis (tip is up) showing persistence of nuclear Cyclin A in clones (white arrows) past the meiotic region and its eventual degradation (yellow arrow). The position of the most advanced Cyclin A-containing heterozygous spermatocytes (located in a different focal plane) is outlined (merged panel). (C,D) In contrast to heterozygotes (C, arrow), in ofs/Df(3R)mbc-R1 mutants, Cyclin A (green) does not overlap with Boule (red) expression (D, line). Notice that ofs/Df has a slightly different (perhaps stronger) Cyclin A phenotype in which all spermatocyte staining is nuclear. (E,F) twine-lacZ reporter, X-Gal activity staining. (E) In heterozygotes (boxed area magnified on right) expression first appears faintly in nuclei of mature primary spermatocytes (arrow), becomes stronger during the meiotic divisions and is strongest in spermatids (arrowhead). Asterisk labels nonspecific, endogenous enzymatic activity. (F) In ofs mutants, expression is first detected relatively more distal in the testis tube (arrow) and resembles the mature primary spermatocyte signal.

 

Figure 4
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  		Fig. 4. ofs spermatocytes activate an abnormal Cyclin A pattern due overexpression of Rux. Whole-mount immunofluorescent images of siblings from a single cross. (A,B) Rux+ progeny show nuclear Cyclin A (CycA, green) co-localized with Rux (red) accumulation in ofs mutants (B, arrow), but not in their heterozygous siblings (A, arrowhead). (C) Siblings heterozygous for ofs still show Cyclin A nuclear translocation in the absence of rux function (arrowhead). (D) ofs mutants inheriting the rux9 mutation have only cytoplasmic localization of Cyclin A (arrow).

 

Figure 5
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  		Fig. 5. ofs encodes the main eIF4G in spermatocytes. (A) Map of the genomic sequence of a portion of the right arm of Chromosome 3 indicating breakpoints of informative deletions (dark gray) and the Z3-3283 point mutation relative to transcripts (red, oriented 5' to 3') and coding regions (blue) for both CG10192 (Ofs) and CG33111. Also shown: in situ probes (light gray), insertion sites for nearby transposable elements (red circles) and flanking genes (green). (B) Predicted Ofs protein and Drosophila eIF4G. Locations are noted for the alternate splice (red), the premature stop in Z3-3283 (asterisk), predicted eIF4E-binding sites (green) and homologous regions (gray). Conserved domains are 27% identical and 43% similar, and include the predicted eIF4A-binding domains. (C-D') In situ hybridizations to wild-type (wt) testes. Tips are marked with asterisks. (C) Antisense probe for ofs (see panel A) showed expression in spermatocytes. (D) Antisense probe to eIF4G showed low expression at the testis tip, and was undetectable thereafter. By contrast, somatic tissue stained strongly (arrows). (C',D') Sense probe controls.

 

Figure 6
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  		Fig. 6. Ofs associates with an mRNA cap analog. (A,B) Western blots of S2R+ cells transfected with Flag-Ofs or Flag-eIF4G (A) or with GFP-Ofs (B) and incubated with either 7-m-GTP-Sepharose (7-methyl-GTP Sepharose) beads or glutathione-Sepharose beads (control). Relevant bands are indicated with an arrow (Ofs constructs) or arrowhead (Flag-eIF4G). Asterisks indicate cross-reacting bands. Input lanes represent 2.5% of total lysate; pulldown lanes represent 100% of protein recovered from the beads. (A) Both Flag-Ofs and Flag-eIF4G are pulled down along with eIF4E by 7-m-GTP-Sepharose beads but not by control beads. (B) Binding is not due to the Flag epitope, because GFP-tagged Ofs also associated with 7-m-GTP-Sepharose beads. All conditions were repeated at least three times with comparable results.

 

Figure 7
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  		Fig. 7. Ofs has an eIF4G-like function in S2R+ cells. (A) Growth curves representing the average counts of GFP-(untransfected) cells from 10-13 wells with error bars representing s.e.m. (B) Western blot against eIF4G and GFP-Ofs assessing the degree of knockdown at days 4 and 7. The major eIF4G band is indicated with an arrow. We variably saw a second band migrating slightly faster (asterisk); its identity is unclear. (C) Matched growth curves for both GFP-(untransfected) and GFP+ (transfected) cells from wells transfected with GFP alone, GFP-Ofs or GFP-eIF4G. Curves were normalized for transfection efficiency at day 3. Key in A applies. Expression of GFP-Ofs or GFP-eIF4G completely rescues the defects caused by eIF4G or eIF4G+Ofs knockdown, but not by eIF4A knockdown. Each curve represents average counts from 3-6 wells, with error bars indicating s.e.m. (D) Cell cycle profiles for the cells represented in A showing a shift towards G1 with either eIF4G or eIF4G+Ofs knockdown. eIF4A knockdown caused an increase in the percentage of cells with sub-G1 DNA content, normally indicative of apoptotic cells (Oancea et al., 2006Go). (E) Summary of cell cycle profiling showing rescue by GFP-Ofs and GFP-eIF4G, but not by GFP alone. Gray bars (GFP-) and green bars (GFP+) are paired for each experimental condition. Within each set, from left to right: sub-G1, G1, S and G2. Notice the relative shifts in the G1 and G2 bars: depletion of eIF4G-like proteins caused an increase in the proportion of G1 cells (compare G1 with G2 bars), and this trend was reversed by transfection of either GFP-Ofs or GFP-eIF4G.

 

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© The Company of Biologists Ltd 2007