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First published online 12 October 2005
doi: 10.1242/dev.02060

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A conserved RNA-protein complex component involved in physiological germline apoptosis regulation in C. elegans

¹ Joslin Diabetes Center and Department of Pathology, Harvard Medical School, One Joslin Place, Boston, MA 02215, USA
² Laboratory for Germline Development, RIKEN Center for Developmental Biology, Kobe, Hyogo 650-0047, Japan

View larger version (22K): [in a new window]   Fig. 1. A C. elegans adult hermaphrodite gonad arm, within which germ cells develop in an assembly-line fashion (Hubbard and Greenstein, 2000; Schedl, 1997). The somatic distal tip cell maintains a population of self-renewing germline stem cells. The first germ cells to enter meiosis form approximately 160 sperm during the L4 stage, then, during adulthood, exclusively oocytes are produced. As germ cells move away from the distal tip, they enter meiotic prophase I in the transition zone, then progress to the pachytene stage. The germline initially consists of a syncytium, in which germ cells are only partially enclosed in a membrane and share a common cytoplasmic core. The gonad bend or `loop' region, where germ cell deaths normally occur, is indicated by a line outside of the gonad. After exit from pachytene, developing oocytes begin to form discrete cells that enlarge as they move through the loop. During diakinesis, a signal from sperm directs oocytes to undergo maturation. Oocytes are then fertilized as they move through the spermatheca into the uterus.

View larger version (34K): [in a new window]   Fig. 2. CAR-1 and CGH-1 associate together in a RNA-dependent complex. (A) Immunoprecipitation of endogenous CGH-1-associated proteins from one-day-old adult hermaphrodites, identified by mass spectroscopy. (B) Each CAR-1 ortholog includes a Sm-like domain within a characteristic conserved region (black) and a variable number of Arginine-Glycine-Glycine (RGG) triplets or functional equivalents (vertical bars) (Birney et al., 1993). An additional conserved region (shaded) contains a FDF domain, a sequence motif of unknown function (Anantharaman and Aravind, 2004). The percentage identity (Id) and similarity (Sim) to C. elegans CAR-1 within these regions was determined by pair-wise BLAST. CAR-1, C. elegans (NP_493254); Hs, H. sapiens (NP_056393); Tral, D. melanogaster (AAF49905); Scd6p, S. cerevisiae (NP_015454). (C) Interaction between CAR-1 and CGH-1 requires RNA. Co-immunoprecipitation of CAR-1 and CGH-1 from C. elegans extracts (lanes 2 and 6) was abolished by RNase A treatment (lanes 3 and 7). Lanes 4 and 8 show control incubations. (D) CAR-1 and CGH-1 are detected specifically in the germline. Lysates from one-day-old adult wild-type worms (N2) and germline-deficient glp-4(bn2) hermaphrodites (Beanan and Strome, 1992) were analyzed by western blotting with CAR-1 and CGH-1 antisera, using Tubulin as a control. The CAR-1 antibody recognized a single germline-specific species of 45 kDa, larger than the predicted size of 37.6 kDa. (E) Diminished CAR-1 protein levels in car-1(RNAi) worms. Protein extracts from wild-type (N2) or car-1(RNAi) one-day-old adults were analyzed by western blotting.

View larger version (101K): [in a new window]   Fig. 3. CAR-1 localization in the germline. Extruded gonads from one-day-old hermaphrodites were analyzed by immunostaining for CAR-1 (A) and DAPI staining for DNA (B). The distal region is to the bottom left and the proximal region at the top right. CAR-1 levels are low in the distal mitotic zone but increase as the germ cells enter meiosis (see detail). (C-J) CAR-1 localization during the pachytene stage. Extruded gonads were stained for CAR-1, CGH-1 and PGL-1. (C) Perinuclear localization of CAR-1; (D) merge of CAR-1 with CGH-1 and DAPI staining. (E-G) Merged CAR-1 (green) and CGH-1 (red) staining (E); merged CAR-1 (green) and PGL-1 (red) staining (F); PGL-1 staining alone (G); images are from the area surrounding the nucleus that is indicated in D. The extent of overlap between CGH-1, CAR-1 and PGL-1 staining was reproducibly comparable to that shown, but among individual foci the degree of overlap and the relative orientation of CGH-1- and CAR-1-stained foci varied. (H-J) A cross-section through the germline core, shown to highlight cytoplasmic CAR-1 (H) and CGH-1 (I) foci; a merged image with DAPI staining is shown in J. (K-N) Specific mislocalization of CAR-1 in the cgh-1(ok492) gonad, revealed by antibody staining. In the mitotic region (K), CAR-1 is present at low levels in perinuclear foci that colocalize with PGL-1 (L), as in wild type (not shown), but its localization becomes dramatically altered within the transition zone (TZ). (K-N) DAPI staining; (K-M) CAR-1 staining (green); (L) merge, including PGL-1 (red), which corresponds to the boxed region in K. Within the pachytene region of cgh-1(ok492) germlines, CAR-1 localization is highly abnormal (M), but the levels and localization of PGL-1 antibody staining are not detectably altered (N). (C-L,N) Single plane confocal images; (M) a confocal z-series projection.

View larger version (71K): [in a new window]   Fig. 4. CAR-1 colocalizes with CGH-1 in the embryo. Two-cell (A-C), four-cell (D-F), 16-cell (G-I), 50-cell (J-L) and 100-cell (M-O) embryos were immunostained for CAR-1 (A,D,G,J,M) and CGH-1 (B,E,H,K,N). Merged images that include DAPI staining are also shown (C,F,I,L,O). Anterior is to the left.

View larger version (94K): [in a new window]   Fig. 5. Drosophila Tral and Me31B form a RNA-dependent complex and colocalize in the germline. (A) RNA-dependent interaction between Drosophila Tral (CAR-1 ortholog) and Me31B (CGH-1 ortholog). Extracts from Drosophila ovaries were immunoprecipitated with a Me31B antibody (Nakamura et al., 2004), and analyzed for the presence of Tral by western blotting. Co-immunoprecipitation of Tral with Me31B (lane 3) was abolished by RNase A treatment (lane 4). Lane 2 shows protein extract incubated with control IgG. (B-D) Extensive colocalization (D) of Me31B (B) and Tral (C) in cytoplasmic granules in the Drosophila germline. A stage-7 egg chamber expressing GFP-Me31B was stained with a Tral-specific antibody.

View larger version (60K): [in a new window]   Fig. 6. Increased physiological apoptosis in car-1(RNAi) hermaphrodites. (A) The number of germ cells that stained with AO was counted in one gonad arm per animal (n>25) at 24 and 48 hours after the L4 molt. This cell death was not observed in the ced-3(n717) background and occurred at similar levels in car-1(RNAi) and ced-9(n1950gf);car-1(RNAi) hermaphrodites. Error bars represent one standard deviation; asterisks denote P<0.05 by t-test. (B) Merged Nomarski and AO staining images of representative wild-type (N2) and car-1(RNAi)animals. AO-positive cells appear to vary in size because they are detected at different stages of death. White arrowheads indicate some of the apoptotic cells. (C) Presence of anucleate cytoplasmic spheres (ACS) in the car-1(RNAi) hermaphrodite gonad. A late one-day-old gonad is shown. White arrowheads indicate ACS, which accumulate in the proximal gonad and have a granular appearance similar to oocytes. Oocytes at the –1, –2 and –3 positions are indicated.

View larger version (100K): [in a new window]   Fig. 7. Enhancement of oogenesis by physiological apoptosis. (A) Apoptosis facilitates progeny production in car-1(RNAi) and cpb-3(RNAi) hermaphrodites. Brood size was only modestly reduced by RNAi depletion of either car-1 or cpb-3 in the N2 background, but was dramatically decreased when RNAi was performed in ced-3(n717) animals. In this representative experiment, N2, n=10; cpb-3(RNAi), n=21; car-1(RNAi), n=19; ced-3, n=10; ced-3;cpb-3(RNAi), n=20; ced-3;cpb-3(RNAi), n=21. Among multiple experiments, the brood size of ced-3(n717) varied between 92% and 98% of wild type, always within the range of statistical insignificance. Importantly, in each experiment car-1 and cpb-3 RNAi resulted in consistent reductions in brood size in the wild-type and ced-3 backgrounds. Error bars represent one standard deviation; asterisks denote P<0.05 by t-test comparing the results of car-1 or cpb-3 RNAi depletion with either N2 or ced-3(n717) controls. (B-F) Nomarski images of ced-3 and RNAi hermaphrodites. (B) One-day-old ced-3(n717);car-1(RNAi) hermaphrodites accumulate abnormal oocytes at the proximal gonad end. DAPI staining (insert) reveals that these proximal oocytes are arrested in diakinesis. A white bar indicates the approximate region from which the DAPI image was obtained from a fixed whole animal. (C) A one-day-old ced-3(n717) hermaphrodite gonad, depicted as in B, is indistinguishable from wild type. (D) A four-day-old ced-3(n717) hermaphrodite gonad. Compared with in the gonad in C, the pachytene region extends further proximally. Note that only three oocytes are present. (E) One-day-old ced-3(n717);cpb-3(RNAi) hermaphrodite gonads have an extended pachytene region (see DAPI staining insert) and fewer oocytes than normal. (F) A three-day-old ced-3(n717);cpb-3(RNAi) hermaphrodite gonad. The extended pachytene region is maintained but abnormal oocytes accumulate proximally, many of which are in diakinesis (not shown). Oocytes are outlined by dashed lines in B,D-F.

View larger version (72K): [in a new window]   Fig. 8. Cytokinesis defects in car-1(RNAi) embryos. Selected frames from a time-lapse Nomarski video recording (not shown) obtained from wild-type (A-H) and car-1(RNAi) (I-P) embryos. The relative time of each exposure is indicted in minutes. Anterior is to the left; black dots represent centrosomes; black arrowheads indicate cleavage furrows; white arrows indicate nuclei in the car-1(RNAi) embryo. Pronuclei appear in the anterior (maternal) and posterior (paternal) regions of the embryo (A,I). The maternal pronucleus migrates to the posterior of the embryo and associates with the paternal pronucleus (B,J). Pronuclei move towards the middle of the embryo before fusing (C,K). Wild-type embryos then initiate the first mitotic division: (C) late prophase; (D) anaphase; (E) appearance of cleavage furrows (arrowheads). The cell cycle continues, producing embryos of two (F), four (G) and six (H) cells. Defects in car-1(RNAi) embryos become apparent during the first cell division. Pronuclear fusion is delayed (K), and cleavage furrows begin to form (not shown) but subsequently regresses, giving rise to a one-cell-embryo containing two nuclei (N). During the next cell cycle cytokinesis is re-initiated and cleavage furrows develop (arrowheads, O), but again these regress coincident with the reappearance of the nuclei. Embryonic cell cycles that are coupled with abnormal cytokinesis continue, resulting in multinucleated cells (P; arrowheads, nuclei).