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First published online 4 July 2007
doi: 10.1242/dev.003764

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Translational control of meiotic cell cycle progression and spermatid differentiation in male germ cells by a novel eIF4G homolog

Departments of Developmental Biology and Genetics, Stanford University School of Medicine, Stanford, CA 94305-5329, USA.

View larger version (28K): [in this window] [in a new window]   Fig. 1. eIF4G2 encodes a novel eIF4G homolog in Drosophila.(A) Alignments of Drosophila eIF4G and eIF4G2 proteins with human eIF4GI (EIF4G1-HUGO) and eIF4GII (EIF4G3-HUGO). Black sections indicate recognized domains, with percentage identical to the eIF4GI middle domain marked. (B) Myc-eIF4E1 co-immunoprecipitates with HA-eIF4G2 from S2 cells. Immunoprecipitation with anti-HA. Western blot probed with anti-Myc and anti-HA, as indicated. Crude extract is 1/10 of each pre-immunoprecipitation (IP) sample.

View larger version (112K): [in this window] [in a new window]   Fig. 2. Loss-of-function mutations in eIF4G2 result in male sterility. (A) Diagram of eIF4G2 intron/exon structure and four strong loss-of-function alleles. Gray, protein coding sequence; black, untranslated regions; diagonal lines, region encoding conserved middle domain. (B) Apical third of a wild-type testis. (C) Apical third of a testis from an eIF4G2 homozygote. (D) Part of testis from eIF4G2 homozygote. (E) Part of testis from eIF4G2 homozygote carrying a single copy of the genomic rescue transgene. eIF4G2 mutant flies were eIF4G2BR21-37/eIF4G2Z3-3283. Scale bars: 100 µm.

View larger version (46K): [in this window] [in a new window]   Fig. 3. eIF4G2 is expressed in a stage-specific pattern within the testis. (A-D) In situ hybridization on wild-type testes; (A) eIF4G2 antisense, (B) eIF4G2 sense, (C) eIF4G antisense, (D) eIF4G sense. (E,F) Anti-HA immunostaining on testes from flies carrying the HA-eIF4G2-rescuing transgene. (E) Spermatocytes (arrow) and elongated spermatids (arrowhead). (F) Early post-meiotic spermatids (arrowhead). (G) Anti-HA staining on a non-transgenic testis (yw). Scale bars: 100 µm.

View larger version (118K): [in this window] [in a new window]   Fig. 4. eIF4G2 is required in the testis for meiotic division. Live squashes of wild-type and mutant germ cells undergoing meiosis. (A,C,E,G,I) Phase contrast. (B,D,F,H,J) Hoechst staining. (A,B) Wild-type and (C,D) eIF4G2 mature spermatocytes. (E,F) Wild-type spermatocytes with condensed chromosomes. (G,H) eIF4G2 spermatocytes with partially condensed chromosomes. (I,J) Wild-type cells in metaphase (arrows) and anaphase (arrowheads). All images are at the same magnification. Scale bar: 20 µm. eIF4G2 mutant flies were eIF4G2BR21-37/eIF4G2Z3-3283.

View larger version (47K): [in this window] [in a new window]   Fig. 5. eIF4G2 is required for translation of twine and cycB in spermatocytes. (A,B) Anti-CycB immunofluorescence on (A) wild-type and (B) eIF4G2 mutant testes. Bracket indicates expression of CycB in mature spermatocytes in wild type. (C,D) X-gal staining of (C) wild-type and (D) eIF4G2 mutant testes expressing a twine-lacZ reporter. (E,F) In situ hybridization on wild-type (E) and eIF4G2 (F) testes with cycB antisense probe. (G,H) In situ hybridization on wild-type (G) and eIF4G2 (H) testes with twine antisense probe. Scale bars: 100 µm. eIF4G2 mutant flies were eIF4G2BR21-37/eIF4G2Z3-3283.

View larger version (137K): [in this window] [in a new window]   Fig. 6. eIF4G2 is required for proper spermatid differentiation.Live squashes of wild-type and mutant germ cells initiating spermatid differentiation. (A,C,E,G,I,K) Phase contrast. (B,D,F,H,J,L) Hoechst staining. (A,B) Wild-type early round spermatids with haploid nuclei (arrow) and mitochondrial derivative (arrowhead). (C,D) Elongating spermatids in wild type. (E,F) Late-stage eIF4G2 cells with large nuclei (arrow) and aggregating mitochondria (arrowhead). (G,H) Terminal cells in eIF4G2 with aggregated mitochondria (arrowhead), large nuclei (arrow) and aberrant partial cellular elongation. (I,J) twine early spermatids. (K,L) twine elongating spermatids. All images are at the same magnification. Scale bar: 20 µm. eIF4G2 mutant flies were eIF4G2BR21-37/eIF4G2Z3-3283. twine mutant flies were tweHB5/tweK08310.

View larger version (74K): [in this window] [in a new window]   Fig. 7. eIF4G2 is not required for the translation of the spermatid differentiation markers fzo and dj. (A-D) Anti-tubulin immunostaining of squashed preparations of (A,B) wild-type and (C,D) eIF4G2 testes. (A,C) DAPI; (B,D) anti-Tub. (E-H) Anti-Fzo immunostaining (F,H) and DAPI (E,G) on squashed preparations of (E,F) wild-type and (G,H) eIF4G2 testes. Arrows indicate location of nuclei; arrowheads mark mitochondrial derivatives (F) or mitochondrial aggregates (H). (I,J) Dj-GFP reporter in (I) wild-type and (J) eIF4G2 testes. Arrowheads indicate Dj-GFP in J. Scale bars: 20 µm. eIF4G2 mutant flies were eIF4G2BR21-37/eIF4G2Z3-3283.

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