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First published online 1 September 2004
doi: 10.1242/dev.01352

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ATX-2, the C. elegans ortholog of ataxin 2, functions in translational regulation in the germline

¹ Division of Basic Sciences, Fred Hutchinson Cancer Research Center, Seattle, WA 98109, USA
² Howard Hughes Medical Institute, Seattle, WA 98109, USA
³ Department of Zoology, University of Washington, Seattle, WA 98195, USA

View larger version (53K): [in a new window]   Fig. 1. ATX-2 forms a complex with a poly(A)-binding protein, and is required for the proper size of the gonad and the onset of oogenesis. (A) A wild-type gonad arm stained with DAPI. Boxed area in A includes sperm (arrow) in the spermatheca. Asterisks in all figures indicate the distal tip of a gonad arm. (B) High magnification of boxed region in A showing the small, highly condensed sperm nuclei (arrowheads). (C) atx-2(RNAi) gonad stained with DAPI; note that the proximal gonad contains only sperm. (D) An enlarged view of some sperm nuclei (arrowheads) from the gonad shown in C. (E) Same gonad as in C stained with mAb1CB4 to visualize spermatocytes and spermatids. (F) Western blot of extracts from wild-type worms (lane 1) and transgenic worms (lane 2) carrying a triple-myc-tagged allele of atx-2 (atx-2::3xmyc); the blot was probed with anti-ATX-2 mAbP1G12. (G) Western blot of extracts from wild-type worms exposed to bacterially expressed atx-2 dsRNA (lane 2) or empty vector control (lane 1). (H,I) Wild-type gonad (H, inset) and atx-2(RNAi) gonad (I) stained for ATX-2; arrows indicate non-gonadal tissues present in the preparation. (J) Left panel: a silver stained SDS-PAGE gel of ATX-2 immunoprecipitates prepared from wild-type worm extracts with either anti-ATX-2 antibodies (mAbP1G12 in lane 1; mAbP6D2 in lane 2) or anti-MEX-5 antibody (lane 3, negative control). (J) Right panel: ATX-2 immunoprecipitates from extracts of wild-type (lane 4) or atx-2(RNAi) worms (lane 5) prepared with anti-ATX-2 mAbP1G12; there is preferential depletion of the lower ATX-2 band. The immunoglobulin heavy and light chains are indicated by asterisks. (K) High-magnification light micrograph of the vulva and gonad of a pab-1(RNAi) animal. The vulva (arrowhead) is protruding from the body, and the entire gonad consists of only a few cells (asterisks mark both distal ends of the gonad). Scale bars: 50 µm.

View larger version (82K): [in a new window]   Fig. 5. The yolk receptor RME-2 is expressed abnormally in ATX-2-depleted gonads. (A-D) Gonads from animals with the indicated genotypes stained for RME-2. (E) Distal region of a wild-type gonad stained for MEX-3. (F) Distal region of a mex-3(RNAi) gonad showing inappropriate expression of RME-2. (G) Composite image showing the distal regions of gonads from animals with indicated genotypes after in situ hybridization for rme-2 mRNA. Only a low level of hybridization is present in the distalmost region (brackets) of the wild-type or mex-3 gonad. (H,I) Central region of gonad from a glp-1(gf);mex-3(RNAi) animal after staining for RME-2 (H) and DAPI (I). Scale bars: 50 µm.

View larger version (36K): [in a new window]   Fig. 2. GLD-1-dependent repression of GLP-1/Notch results in underproliferation of germ cells in ATX-2-depleted gonads. (A) The mean number of phosphorylated-H3-positive, mitotic nuclei per gonad arm (n=number of gonad arms)±mean deviation, was determined for animals with the indicated genotypes. The fem-3(gf) and fem-1 mutations masculinize and feminize the germline, respectively. Depletion of GLD-1 restored the number of mitoses in gld-1;atx-2(RNAi) gonads to the wild-type level. (B-G) Magnified images of the distal gonad after staining for GLP-1 (B-D) or GLD-1 (E-G). Abnormally high levels of GLD-1 are present in the distalmost part of masculinized atx-2(RNAi) gonads (F), but not in many feminized, fem-1;atx-2(RNAi) gonads as shown here (G). Scale bars: 50 µm.

View larger version (80K): [in a new window]   Fig. 3. Masculinization of ATX-2-depleted germlines is caused by continuous repression of tra-2 mRNA translation. (A-C) tra-2(gf);atx-2(RNAi) gonad after staining with DAPI (A,B) and mAb1CB4 (C). Sperm are absent (C, compare with Fig. 1E) and the oocyte-like germ cells are present (boxed region in A, magnified in B). Each arrowhead in B, and in the wild-type control shown in D, indicates a single oocyte nucleus with multiple highly condensed chromosomes (irregular bar-like objects). (E,F) gld-1;atx-2(RNAi) gonad showing the presence of sperm. Boxed area in E is shown at higher magnification in F; sperm nuclei (arrowheads) are small and highly condensed. Some gld-1;atx-2(RNAi) germlines showed mitotic proliferation proximal to the sperm; this phenotype was also observed in male or masculinized hermaphrodite germlines depleted of GLD-1 at 25°C. (G-I) Nomarski micrographs of fog-2 and fog-2;atx-2(RNAi) animals with the gonads outlined in black. The gonads of most fog-2;atx-2(RNAi) animals, as shown here, were small and contained only oocytes similar to those in fog-2 gonads (arrows). Boxed area in H is shown at higher magnification in I; the spermatheca (arrowhead) does not contain any sperm. A minority of fog-2;atx-2(RNAi) animals contained gonads with either both sperm and oocytes, or only sperm. Scale bars: 50 µm.

View larger version (101K): [in a new window]   Fig. 4. Germ cells fail to enter pachytene in gld-2;atx-2(RNAi) gonads. Dissected gonads after staining for HIM-3 or with DAPI; insets show magnified views of nuclei from regions marked with arrowheads. Photographs in A and B were taken with identical exposures, as were photographs in D and F. Pachytene chromosomes in A-D have a characteristic thread-like appearance. The chromosomes of gld-2;atx-2(RNAi) germ cells did not resemble normal pachytene nuclei in morphology when stained with DAPI (E) or in HIM-3 localization (F). Approximately half of the gld-2;atx-2(RNAi) gonads analyzed showed inappropriate mitotic proliferation in the proximal germline (region indicated by bent arrow in E,F). Scale bars: 50 µm.

View larger version (74K): [in a new window]   Fig. 6. GLD-1 restricts MEX-3 expression to the distalmost germ cells, where MEX-3 represses translation of RME-2. (A) Control heterozygous gld-1/+ animal showing MEX-3 restricted to the distalmost, mitotic region, as in wild-type gonads (compare with Fig. 5E). (B) gld-1 mutant gonad showing MEX-3 in both the distal and medial regions of the gonad. In tumorous gld-1 gonads, germ cells that leave the distalmost region enter pachytene and then re-enter mitosis. MEX-3 is expressed throughout the gld-1 germline, including in the pachytene cells, suggesting that its broad expression is not due simply to re-entry of germ cells into mitosis. Similarly, gld-1;atx-2(RNAi) gonads are not tumorous and express MEX-3 inappropriately in meiotic germ cells (D). (E) Model showing rme-2 mRNA at very low levels (gray) in the distalmost gonad and increasingly high levels (black) in the medial gonad. GLD-1 restricts MEX-3 expression to the distalmost gonad, where MEX-3 prevents the low levels of rme-2 mRNA from being translated. (F) RME-2 is translated in the distalmost part of masculinized gld-1;atx-2(RNAi) germline, but not in the control fem-3(gf) masculinized germline. Removal of GLD-1 alone from either male germlines or from masculinized hermaphrodite germlines, does not result in the distalmost expression of RME-2. Depletion of GLD-1 from atx-2(RNAi) germline restores the distalmost, mitotic region of the germline. (G) Masculinized germlines of gld-1;atx-2(RNAi) and fem-3(gf) animals have similar, low levels of rme-2 mRNA in the distalmost germline (brackets). (H-J) Gonad from a glp-1(gf);atx-2(RNAi) animal stained with DAPI (H), for MEX-3 (I) and RME-2 (J). MEX-3 is present throughout the gonad, but is unable to repress RME-2. Scale bars: 50 µm.