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cgh-1, a conserved predicted RNA helicase required for gametogenesis and protection from physiological germline apoptosis in C. elegans

Rosa E. Navarro1, Eun Yong Shim1,*, Yuji Kohara3, Andrew Singson2 and T. Keith Blackwell1,{ddagger}

1 Center for Blood Research and Department of Pathology, Harvard Medical School, 200 Longwood Avenue, Boston, MA 02115, USA
2 Waksman Institute, Rutgers University, Piscataway, NJ 08854, USA
3 Japan Science and Technology Corporation, Genome Biology Laboratory, National Institute of Genetics, Mishima 411-8540, Japan
* This author made an important independent contribution to this work



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  		Fig. 1. Structure and conservation of cgh-1. (A) cgh-1 gene structure. The predicted cgh-1 open reading frame (C07H6.5) of 1290 bp contains four exons (black boxes, to scale) and three introns (not to scale). A 3' UTR of approximately 900 bp (not to scale) is predicted by an AAUAAA motif and by cDNA sequences (Y. K., not shown). (B) Conservation of CGH-1. The predicted C. elegans CGH-1 protein is compared with Drosophila ME31B (AAF52881), human RCK (P26196), S. pombe Ste13 (BAA06178), S. cerevisiae DhhIp (NP_010121), a human eIF4-A isoform (NP_001407) and the closest related C. elegans eIF4-A ortholog (F57B9.6). CGH-1 orthologs are designated by a bracket. In each protein a white central box represents the conserved helicase region, with its percentage identity to CGH-1 indicated. Gray boxes indicate less conserved regions, and black boxes shown only in CGH-1 correspond to homology motifs, which are characteristic of RNA helicases (de la Cruz et al., 1999).

 


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  		Fig. 2. Structure of the C. elegans hermaphrodite germline and expression of cgh-1 mRNA. (A) Diagram of the hermaphrodite germline. As described, two gonad arms each produce approximately 1000 germ cells (Schedl, 1997). A somatic distal tip cell (DTC) maintains germ cell proliferation. As germ cells enter meiotic prophase, a central gonad core appears that is more prominent in hermaphrodites. This common core disappears as oocyte precursors cellularize and progress from the pachytene to the diakinesis stage of meiosis. Prior to fertilization in the spermatheca each oocyte undergoes maturation, including breakdown of the nuclear envelope. D, distal, L, loop, P, proximal, O, oocytes; S, spermatheca; E, embryo and U, uterus. (B) Presence of cgh-1 mRNA in germline mutant strains. Total RNA from synchronized cultures grown at the restrictive temperature was extracted from the wild type (N2) and indicated strains. fem-1(hc17) produce only oocytes, fem-3(q20gf) produce only sperm, and in glp-4(bn2) the germ line is underproliferated. RNA was blotted and hybridized to a full-length cgh-1 cDNA. Expression levels were normalized to a ribosomal protein gene, rpp-1, and indicated at the bottom. (C-F) cgh-1 expression assayed by in situ hybridization. Hybridization was performed on wild type synchronized worms using the cgh-1 cDNA clone yk85e1 (DDBJ/GenBank accession number D74793) as a probe. (C) L1 larval stage. Low levels of expression are detected in the germ cells Z2 and Z3. (D) L2/L3. (E) Early L4 and (F) young adult. h, head; l, loop; p, proximal; d, distal; v, vulva.

 


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  		Fig. 3. Presence of CGH-1 in adult hermaphrodite gonads. (A-K) Extruded gonads from one-day old wild type (A-I) and cgh-1(RNAi) (J and K) hermaphrodites were stained with affinity purified rat CGH-1 antibody (A,D,G,J), an antibody against the constitutive P granule component PGL-1 (B,E,H) (Kawasaki et al., 1998), and the DNA dye DAPI (K), then examined by confocal (A-I) or fluorescence microscopy (J,K). C,F and I are merged images of CGH-1 and PGL-1 staining. (A-C) Individual section through the gonad center that highlights CGH-1 staining in the core region. The distal tip is at top left. No CGH-1 staining was visible in the spermatheca, which is to the bottom left and not shown. Nuclei that stain with DAPI are located in the center of the P granule rings (not shown). (D-F) Detail of the surface of a different gonad, immediately distal to the loop area. (G-I) A section through the center of oocytes. Arrowheads in G indicate some of the CGH-1 staining that colocalizes with PGL-1. Examination of multiple focal planes revealed that in oocytes many PGL-1 particles were larger than the corresponding CGH-1 staining area, but also that in general a discrete focus of CGH-1 staining was co-localized with each P granule (not shown). For example, the very large P granule located to the lower right of the star in H was co-localized with a CGH-1 particle in an adjacent focal plane. Only diffuse background CGH-1 staining is detected in cgh-1(RNAi) gonads (J).

 


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  		Fig. 4. CGH-1 expression in adult male gonads. Extruded male wild type (A-D) and cgh-1(RNAi) (E,F) gonads were stained with anti-CGH-1 (A and E), anti-PGL-1 (B), DAPI (D,F), and examined by fluorescence microscopy. A merged image of A, B and D is shown in C. In each panel, an enlargement of the boxed area is shown. CGH-1 staining is less prominent in male gonads than in hermaphrodites, necessitating longer exposure times. In addition to the granular staining detailed in A, these exposures reveal a diffuse pattern, which may represent background. The granular CGH-1 staining is not observed in cgh-1 (RNAi) male gonads (E). d, distal and p, proximal.

 


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  		Fig. 5. Presence of CGH-1 in the embryo. Embryos of the 2-cell (A-D), late 4-cell (E-H), approximately 50-cell (I-L) and approximately 100-cell (M-P) stages were stained with anti-CGH-1 (A,E,I,M), anti-PGL-1 (B,F,J,N) and DAPI (D,H,L,P) and examined by fluorescence microscopy. C, G, K and O are merged images. Examination of four-cell embryos by confocal microscopy indicated that, in general, each PGL-1 particle co-localized with a CGH-1 particle (not shown).

 


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  		Fig. 6. Effects of cgh-1 RNAi on oogenesis and spermatogenesis. (A and B) Nomarski DIC views of gonads within 1-day-old wild type (A) and cgh-1(RNAi) (B) hermaphrodites. d, distal; l, loop; s, sperm; o, oocytes; do, degraded oocytes and e, embryo. (C-J) Expression of germline proteins in gonads extruded from 1-day-old wild type (C,E,G,I) and cgh-1(RNAi) (D,F,H,J) hermaphrodites. Gonads were stained with antibodies to the NOTCH homolog GLP-1 (Crittenden et al., 1994; C-D), the meiotic protein GLD-1 (Jones et al., 1996; E-F), the yolk receptor RME-2 (Grant and Hirsh, 1999; G-H), and the P granule component PGL-1 (I-J). d, distal; p, proximal. (K, L) Sperm that were prepared from wild-type and cgh-1(RNAi) gonads were induced with pronase to visualize their pseudopods. Boxes surround a detailed view.

 


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  		Fig. 7. Increased germline apoptosis in cgh-1(RNAi) hermaphrodites. (A) Acridine orange (AO) staining. Wild type control (blue) and cgh-1(RNAi) (red) hermaphrodites from the indicated stages were AO-stained, then anesthetized and visualized by fluorescence and Nomarski DIC microscopy (as in C and D). Numbers of AO-positive cells per gonad arm were counted in L4 animals (0 hours) and at the indicated times thereafter, then plotted. (B) Germline cell corpses. Numbers of germline cell corpses per gonad arm were counted in living wild type and cgh-1(RNAi) hermaphrodites, and graphs constructed as in A. (C-J) Merged AO staining and Nomarski images of representative 2-day-old no RNAi control (C,E,G,I) and cgh-1(RNAi) (D,F,H,J) hermaphrodites. Experiments were performed in the following genetic backgrounds: wild type (C,D), ced-3(n717) (E,F), mpk-1(ga117), (G and H) and ced-9(n1950gf) (I,J). White arrowheads indicate representative germline corpses, and orange arrowheads indicate the gonad regions of which Nomarski details are shown in C and D. The diffuse background AO staining apparent in many panels derives from the intestine. d, distal; p, proximal and i, intestine. (K and L) Nomarski views of representative 2-day-old ced-1 (K) and ced-1; cgh-1(RNAi) (L) hermaphrodites, labeled as in C and D. ced-1; cgh-1(RNAi) animals contained increased numbers of typical corpses in the loop region (see inset) along with numerous small unengulfed oocyte corpses located more proximally.

 


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  		Fig. 8. Detailed views of oocytes in 2-day-old ced- 3 and ced-3; cgh-1(RNAi) hermaphrodites. (A,B) Nomarski view of ced-3 (A) and ced-3; cgh-1(RNAi) (B) oocytes. (C-H) Gonads extruded from ced-3 (C,E,G) and ced-3; cgh-1(RNAi) (D,F,H) animals were stained with DAPI (C,D), anti-GLH-1 (E,F) or anti-RME-2 (G,H).

 

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© The Company of Biologists Ltd 2001