Knockdown of eIF4G2 in early stages affects meiotic divisions and differentiation during spermatogenesis.

<p>Wild-type testes (A-A'', C-C'') and testes expressing <i>eIF4G2</i> shRNA under <i>nos</i>-Gal4:VP16 control (B-B'', D-D'') were co-stained with anti-eIF4E-3 (red) and anti-eIF4G (green). Panels A-B'' show entire testes while the distal end of the testes is shown C-D''. The merged images are shown in A''-D''. DAPI staining is shown in magenta. Note the absence of elongated flagellar axonemes and nuclear bundles in the eIF4G2 knockdown testes (D') as compared with the wild-type (C'). Scale bar 100 μm.</p

Distribution of Don Juan-GFP and Orb in <i>eIF4E-1</i> and <i>eIF4E-3</i> knockdown testes.

<p>GFP fluorescence of Don Juan-GFP (DJ-GFP) in the wild-type background (A) or testes expressing <i>eIF4E-1</i> (B) and <i>eIF4E-3</i> (C) shRNA under the <i>bam</i>-Gal4:VP16 driver. Whole mount anti-Orb staining (green) of wild-type (A') or <i>eIF4E-1</i> (B') and <i>eIF4E-3</i> (C') knockdown testes. All knockdowns were performed with shRNA driven by the <i>bam</i>-Gal4:VP16 driver. The testis outline is outlined with a dashed line in A-C. DAPI is shown in magenta. Scale bar 100 μm.</p

Knockdown of <i>eIF4E-1</i> and <i>eIF4E-3</i> affects testes development and spermatid differentiation.

<p>Wild-type testes (A-A''') and testes expressing <i>eIF4E-1</i> shRNA with <i>nos</i>-Gal4:VP16 driver (B-B''') were stained with anti-eIF4E-3 (red, A, B) and anti-eIF4G antibody (green, A', A'' & B', B''). The small rudimentary testes are marked by arrows while the accessory glands are marked by arrowheads (B, B'). A magnified view of the apical tip of <i>eIF4E-1</i> knockdown testes shows a mass of undifferentiated cells surrounded by a disorganised muscle sheath (B'', B''') as compared with the wild-type (A'', A'''). The DNA is stained with DAPI (magenta). Scale bars 100 μm (A, A', B, B') and 50 μm (A''', B'''). Expression of <i>eIF4E-1</i> shRNA using c587-Gal4 driver results in a degenerate testes structure (C', arrows) as revealed by phase contrast microscopy. The arrowheads indicate the accessory glands. The wild-type testis is shown in C. Scale bar 100 μm. (D) The distal end of wild-type testes, stained with anti-eIF4E-1 (green) and anti-eIF4E3 (red) antibody shows well organized haploid nuclear bundles (magenta, arrows). A similar region of a <i>bam</i>-Gal4-<i>eIF4E-1</i> RNAi testis (D', D'') co-stained with anti-eIF4E-1 (green) and anti-eIF4E-3 (red) antibody reveals severe defects in nuclear compaction and individualisation; the post-meiotic nuclei (stained with DAPI, magenta) are found along the elongated spermatids (arrows). Scale bar 100 μm. (D''', D'''') Knockdown of <i>eIF4E-3</i> in spermatocytes using <i>bam</i>-Gal4:VP16 driver results in loss of nuclear condensation and bundling at the distal end of the testes (arrows). eIF4E-3 and eIF4E-1 immunostaining is shown in red and green, respectively. The DNA is stained with DAPI (magenta). Scale bar 100 μm.</p

eIF4G and eIF4G2 act redundantly during early spermatogenesis.

<p>Knockdown of <i>eIF4G</i> and <i>eIF4G2</i> using the <i>nos</i>-Gal4:VP16 driver results in a rudimentary testes structure (marked by arrows). Anti-eIF4E-1 (A) and anti-eIF4E-3 (B) immunostainings are shown in green and red, respectively, while the merged image is shown in C. Absence of eIF4E-3 positive cells indicates spermatogenesis arrest at the spermatogonia stage. The magnified image of the apical tip of the testis (arrow, D, E) shows a mass of cells. DNA is stained with DAPI (magenta). Scale bar 100 μm.</p

eIF4G is required in the somatic cyst cells for normal spermatogenesis.

<p>Wild-type testes (A-C') and testes expressing <i>eIF4G</i> shRNA with c587-Gal4 driver (A''-C''') were co-stained with anti-eIF4G (red) and anti-eIF4E-3 (green) antibodies. Loss of anti-eIF4G staining in the cyst cells at the apical tip of testis (B'') indicates efficient knockdown of the protein. The testis shows an increased number of abnormal germ cell cysts at its apical tip (B'') and reduced meiotic and post-meiotic cysts along its length (compare A' & C' with A''' & C''') as revealed by anti-eIF4E-3 staining (green). Although mature sperm nuclear bundles are found at the distal end of the testes, they appear disorganised (C''', arrows) and nuclei at various stages of condensation are also found at the caudal end of the elongating spermatids (B''', arrows).</p

Effects of <i>eIF4E-1</i> and <i>eIF4G2</i> knockdown on spermatogenesis.

<p>Phase-contrast micrographs of squash preparations (left panel) and the corresponding Hoescht staining (right panel). As compared with the wild-type (A-B'), testes expressing shRNA targeting <i>eIF4E-1</i> (C-D') and <i>eIF4G2</i> (G) in the spermatocytes using <i>bam</i>-Gal4:VP16 driver show defects in nuclear condensation and cytokinesis, while knockdown of <i>eIF4G2</i> using <i>nos</i>-Gal4:VP16 driver (E-F') results in defective cytokinesis. The nuclei in prophase I, anaphase I and onion stage spermatids are marked by arrows in magenta, yellow and red, respectively. The arrowhead (yellow) shows the nebenkern in panels B & B', D & D' and E' & E' while the mitochondrial structure in elongated spermatids and spermatocytes is marked in F & F' and G & G', respectively. Note the three chromosome clumps in B', C' and D'.</p

Distribution of eIF4E-1, eIF4E-3, eIF4G and eIF4G2 in the wild-type testes.

<p>(A) Schematic diagram showing the stages of spermatogenesis in <i>Drosophila</i> testes. The hub cells (HC) at the apical tip of the testes maintain the stem cells (SC) which include a germline stem cell and a somatic cyst stem cell. The germline stem cell differentiates into spermatogonia (SG) which divide mitotically to produce primary spermatocytes (PSC). The spermatocytes undergo rapid cellular growth to form mature spermatocytes (MSC) that undergo meiosis, producing haploid onion stage spermatids (OSS). Following cellular transformation and differentiation, they develop flagella with the nuclei (magenta) at the distal end of the testes. ES = Elongated spermatids. Whole-mount immunostaining of adult testes using antibodies that recognise eIF4E-1 (B-B'', green), eIF4E-3 (C-C'', red), eIF4G (D-D'', green) and eIF4G2 (E-E'', green). The top panel (B-E) shows the entire testes while the middle (B'-E') and lower panels (B''-E'') show the apical and distal end, respectively. DAPI is shown in magenta. Scale bar 100 μm.</p

(A) In a heterozygous control niche, the periphery of each cap cell (CpC) is labeled strongly with antibodies to Armadillo (Arm, red), whereas the terminal filament (TF) is only weakly labeled

(B and C) In mosaic niches, Arm staining is increased between mutant cells. This increase is especially pronounced at the interface between mutant and wild-type TF cells (arrows). Bar, 10 μm.<p><b>Copyright information:</b></p><p>Taken from "RanBPM regulates cell shape, arrangement, and capacity of the female germline stem cell niche in "</p><p></p><p>The Journal of Cell Biology 2008;182(5):963-977.</p><p>Published online 8 Sep 2008</p><p>PMCID:PMC2528568.</p><p></p

(A) Two RanBPM isoforms share a core region with a SPRY domain, LisH/CTLH motifs, and a CRA domain

Long RanBPM has an unstructured N-terminal extension. RanBPM is highly homologous to mouse RanBPM and mouse RanBP10: the percent amino acid identity within the four conserved domains is shown. Protein fragments used to make antibodies are noted. (B) is alternatively spliced to regulate the use of alternate start codons (ATG1–2). Exons (1–12) are colored white for UTRs, black for coding regions, and gray for the alternate coding region. Transposable element insertions: a Gal4 element in the 5′ UTR (), a weak loss of function element insertion in the first intron (), and a strong loss of function piggyBac insertion in exon 11 (). ) is a deletion of the region between the FRT sequences in and , and the N-terminal deletion is a element excision derivative of . (C) Antibodies raised against the N-terminal fragment of long RanBPM (αN-term) recognize a 140-kD band (L, long RanBPM) in heterozygous extracts that is absent from extracts. Antibodies raised against the core domains of RanBPM (α-Core) recognize two protein isoforms: long RanBPM (140 kD) and short RanBPM (67 kD). Several cross-reacting bands are marked with asterisks. (D) Summary of complementation tests between various alleles. Blue boxes indicate complete lethality of the transheterozygote, whereas each number indicates the percentage of surviving transheterozygotes over the number of survivors expected if the alleles had fully complemented. Relative allele strength at 25°C is . is a temperature-sensitive lethal allele, and these tests were performed at the restrictive temperature of 25°C. (E) does not express long RanBPM (L), which is recognized with the N-terminal antibody, but does express short RanBPM (S), which is recognized with the core antibody. was included as a control to identify bands specific to RanBPM.<p><b>Copyright information:</b></p><p>Taken from "RanBPM regulates cell shape, arrangement, and capacity of the female germline stem cell niche in "</p><p></p><p>The Journal of Cell Biology 2008;182(5):963-977.</p><p>Published online 8 Sep 2008</p><p>PMCID:PMC2528568.</p><p></p

(A and B) In mosaics, loss of causes incompletely penetrant defects in epithelial structure, causing egg chamber fusion (A) and follicle cell multilayering, which mainly affects clones positioned at the anterior or posterior poles of the egg chamber (B)

(C and C′) Two partial projections from a single optical stack though a mosaic late stage oocyte. mutant follicle cells are marked by lack of GFP (green) and lack of RanBPM (blue), and nuclei are labeled with DAPI (red). An extended dorsal appendage (arrow) is surrounded by wild-type cells in C, whereas on the other side of the embryo (C′), mutant cells surround a short, paddle-like dorsal appendage (arrow). (D) Homozygotes for the hypomorphic allele are fertile and have normal egg chamber morphology except for an incompletely penetrant defect in oocyte nuclear positioning. The normal posterior position of the oocyte is marked with an arrowhead, and the ectopic position is marked with an arrow. (E and F) The phenotype of is normal except for an oocyte localization defect, but a dominant enhancement after removing one copy of causes egg chamber fusion (E) and loss of epithelial integrity (F). Bars, 25 μm.<p><b>Copyright information:</b></p><p>Taken from "RanBPM regulates cell shape, arrangement, and capacity of the female germline stem cell niche in "</p><p></p><p>The Journal of Cell Biology 2008;182(5):963-977.</p><p>Published online 8 Sep 2008</p><p>PMCID:PMC2528568.</p><p></p