Dynein Light Chain DLC-1 Facilitates the Function of the Germline Cell Fate Regulator GLD-1 in Caenorhabditis elegans

Abstract

Developmental transitions of germ cells are often regulated at the level of post-transcriptional control of gene expression. In the Caenorhabditis elegans germline, stem and progenitor cells exit the proliferative phase and enter meiotic differentiation to form gametes essential for fertility. The RNA binding protein GLD-1 is a cell fate regulator that promotes meiosis and germ cell differentiation during development by binding to and repressing translation of target messenger RNAs. Here, we discovered that some GLD-1 functions are promoted by binding to DLC-1, a small protein that functions as an allosteric regulator of multisubunit protein complexes. We found that DLC-1 is required to regulate a subset of GLD-1 target messenger RNAs and that DLC-1 binding GLD-1 prevents ectopic germ cell proliferation and facilitates gametogenesis in vivo Additionally, our results reveal a new requirement for GLD-1 in the events of oogenesis leading to ovulation. DLC-1 contributes to GLD-1 function independent of its role as a light chain component of the dynein motor. Instead, we propose that DLC-1 promotes assembly of GLD-1 with other binding partners, which facilitates formation of regulatory ribonucleoprotein complexes and may direct GLD-1 target messenger RNA selectivity.

Keywords: RNA binding protein; germline; post-transcriptional regulation; tumor.

Figure 1

(A) Schematic of C. elegans adult germline. The stem and progenitor cell region resides at the distal end followed by the transition zone where cells switch from mitosis to meiosis. Next is the pachytene region where germ cells undergo meiosis and the oocytes are located at the proximal end. (B) The regulatory network that controls the decision between stem cell proliferation and differentiation (mitotic and meiotic cell cycles). GLP-1/Notch signaling activated by a somatic distal tip cell (yellow) promotes mitotic divisions in the stem cell region of the worm germline (blue). FBF-1 and FBF-2 proteins bind and repress mRNAs that would initiate meiosis or differentiation. The GLD-1/NOS-3 and GLD-2/3 pathways promote entry into meiosis (maroon). We hypothesize that DLC-1 (green) promotes GLD-1 function.

Figure 2

Genetic evidence that dlc-1 functions with gld-1. Dissected gonads of the indicated genotypes were stained for the M-phase marker phosphohistone H3 (pink) and DNA (blue). Stem and progenitor cells are detected by anti-REC-8 staining (green). (A) Wild-type control cultured at 20°. Single mutant animals including (B) dlc-1(tm3153), (C) gld-3(q730), (D) gld-2(q497), and (E) nos-3(q650) show no ectopic germ cell proliferation. Aberrant proliferation (white arrows) is observed in (H) gld-2(q497); dlc-1(tm3153) and (G) gld-3(q730); dlc-1(tm3153) double mutant animals (inset depicts a gld-2(q497); dlc-1(tm3153) mutant germline with >35 stem and progenitor cell rows), but not in (F) nos-3(q650); dlc-1(tm3153). No ectopic proliferation is detected in (I) dhc-1(or195) single mutant animal, (J) dhc-1(or195); gld-3(q730) double mutant animal, (K) dhc-1(js121) mutant treated with control RNAi, or (L) dhc-1(js121); gld-2(RNAi). Efficacy of gld-2(RNAi) was determined by scoring sterility and embryonic lethality of dhc-1(js121)/hT2; gld-2(RNAi) treated worms. dhc-1(js121)/hT2; gld-2(RNAi) treated worms exhibited 20 ± 6% sterility and for worms with progeny the embryonic lethality was 98 ± 0.9%. Fluorescence micrographs are representative images of data collected from at least two independent experiments and 19–80 worms were scored for each genotype (see Table 1). Bar, 10 μm.

Figure 3

Length of mitotic region in dlc-1, dhc-1 and gld mutant germlines. To identify defects in the transition from mitosis to meiosis, the number of REC-8–positive stem and progenitor cell rows from the distal end of the germline to the transition zone boundary were counted. A statistically significant increase in the lengths of the distal proliferative zones was observed in gld-2(q497); dlc-1(tm3153) germlines compared to gld-2(q497) control (indicated by **; P = 0.0025 by Kolmogorov–Smirnov test). No significant changes in mitotic zone length were detected in gld-3(q730); dlc-1(tm3153) vs. gld-3(q730) germlines (P > 0.25 by Kolmogorov–Smirnov test). Data were collected from at least two independent experiments and 24–80 germlines were scored for each genotype.

Figure 4

dlc-1(-) synthetic overproliferation phenotype is not dependent on glp-1 activity. (A) Fluorescence micrograph of dissected gonad from triple mutant animal gld-3(q730); dlc-1(tm3153) unc-32(e189) glp-1(q46) immunostained for mitotically dividing cells (pink), stem and progenitor cells (green), and DNA (blue). White arrows indicate aberrant cell proliferation. (B) Fluorescence micrograph of gld-2(q497); dlc-1(tm3153) unc-32(e189) glp-1(q46) whole worm stained with DAPI show the presence of many sperm (white arrows). (C) The ability of gld-2(q497); dlc-1(tm3153) unc-32(e189) glp-1(q46) mutant germ cells to differentiate into sperm is gradually lost after several generations, as seen in a dissected gonad stained with DAPI. (D) dlc-1(tm3153) unc-32(e189) glp-1(q46) whole worm stained with DAPI. White arrow indicates sperm. Bar, 10 μm.

Figure 5

DLC-1 helps regulate a subset of GLD-1 target mRNAs in a dynein motor independent manner. Fluorescence micrographs of dissected gonads expressing GFP-tagged Histone H2B under the control of either (A) cye-1 3′UTR or (B) spn-4 3′UTR after the indicated RNAi treatments. Bar, 10 μm. (C) Quantitation of reporter expression in the meiotic pachytene region of C. elegans germline after the following RNAi treatments: control (gray), dlc-1 (green), gld-1 (yellow), and dhc-1 (blue). Transgenes are identified along the x-axis, and the number of germlines scored (N) is indicated for each treatment. Efficiencies of dlc-1 and dhc-1(RNAi) treatments were established by confirming 100% sterility. Nuclear GFP signal in meiotic pachytene germ cells was quantified using LAS-X software (Leica). Signal intensities following experimental RNAi treatments (dlc-1, gld-1, or dhc-1) were normalized to respective control values. Results are representative of at least three independent experiments and error bars represent SD from the mean. Student’s unpaired t-test was used to calculate P-values for dlc-1 and dhc-1(RNAi) treatments compared to control. * indicates statistically significant differences.

Figure 6

GLD-1 protein expression pattern and abundance is similar in wild-type vs. dlc-1(-) worms. Fluorescence micrographs of (A) wild-type and (B) dlc-1(tm3153) dissected gonads immunostained for GLD-1 protein using anti-GLD-1 antibody (Jones et al. 1996). Bar, 10 μm. (C) Western blot analysis of GLD-1 protein levels in the lysate of 50 wild-type or dlc-1(tm3153) whole worms probed with anti-GLD-1, anti-tubulin, and anti-MYO-3 antibodies. * indicates 210 kDa myosin heavy chain band. (D) Quantitation of GLD-1 protein expression in meiotic pachytene germ cells using LAS-X software (Leica). Wild type, N = 14; dlc-1(tm3153), N = 13; P > 0.17. Quantitation of GLD-1 protein level normalized to (E) tubulin and (F) myosin. Error bars represent SD from the mean. No significant differences in GLD-1 protein levels were observed in dlc-1(-) vs. wild-type control (GLD-1/anti-tubulin: P > 0.7, N = 3 of each genotype; GLD-1/anti-myosin: P > 0.4, N = 3 of each genotype). Student’s unpaired t-test was used to calculate P values.

Figure 7

DLC-1 binds the N-terminal domain of GLD-1 in vitro. (A) Schematic diagram showing the domain structure of GLD-1. GLD-1 forms homodimers through interactions in the QUA1 domain and the KH and QUA2 domains bind RNA. Mutations to the N-terminal domain are indicated. (B) GST pulldown assay to test if GST alone or GST-DLC-1 (detected by Coomassie) binds either wild-type His₆ GLD-1, His₆ GLD-1 that has a mutated DLC-1 binding site (SQT to AAA mutant), or His₆ GLD-1 that has a conserved serine mutated to alanine (S22A mutant). His₆ GLD-1 constructs were detected by Western blot. (C) GST and GST-DLC-1 were assayed for the ability to bind wild-type His₆ GLD-1 in the presence or absence of RNase.

Figure 8

GLD-1^ndb mutation does not affect protein expression and localization in vivo. GLD-1^wt::OLLAS and GLD-1^ndb::OLLAS protein expression (green) in (A) gld-1(q485); gld-1^wt::ollas and (B) gld-1(q485); gld-1^ndb::ollas transgenic worm gonads. GLD-1::OLLAS is highly expressed in the meiotic pachytene region of the germline. DNA was stained using DAPI (gray) for reference. Bar, 10 μm. (C) Quantitation of GLD-1::OLLAS immunostaining intensity levels in meiotic pachytene cells using LAS-X software (Leica). No significant difference in expression was observed using Student’s unpaired t-test (P > 0.6; gld-1^wt::ollas N = 12, gld-1^ndb::ollas N = 13). (D) Western blot analysis of GLD-1^wt::OLLAS and GLD-1^ndb::OLLAS protein levels in whole lysates of transgenic worms. Tubulin is used as a loading control. (E) Quantitation of GLD-1^wt::OLLAS and GLD-1^ndb::OLLAS protein level in 50 transgenic whole worm lysate normalized to tubulin. No significant difference in expression was detected by Student’s unpaired t-test (P > 0.7; N = 5 for each genotype). (F) Confocal images of meiotic pachytene region of wild-type, gld-1(q485); gld-1^wt::ollas, and gld-1(q485); gld-1^ndb::ollas germlines co-immunostained for P granule component PGL-1 (red) and GLD-1::OLLAS (green). Bar, 10 μm. (G) Quantitation of GLD-1, GLD-1^wt::OLLAS and GLD-1^ndb::OLLAS protein enrichment in P granules from confocal images. No significant difference in enrichment of GLD-1^wt::OLLAS and GLD-1^ndb::OLLAS was detected by Student’s unpaired t-test (P > 0.6; N = 3 for each genotype).

Figure 9

DLC-1/GLD-1 interaction promotes GLD-1 function in vivo. (A) Analysis of sterility phenotype of gld-1(q485); gld-1^wt::ollas and gld-1(q485); gld-1^ndb::ollas worms at 24°. Sterile worms were identified by the inability to produce viable offspring. L4 larvae from synchronous cultures of either gld-1(q485); gld-1^wt::ollas or gld-1(q485); gld-1^ndb::ollas worms were isolated and their ability to produce viable offspring assessed. Percent sterility from four independent experiments was calculated and error bars represent SD from the mean. Number of animals scored for each genotype (N) is indicated below the chart. ** indicates statistically significant difference in percent sterility determined by Student’s paired t-test (P = 0.001). (B) Images of gld-1(q485); gld-1^wt::ollas and gld-1(q485); gld-1^ndb::ollas whole worms acquired using Nomarski DIC microscopy. Morphological landmarks include oocytes, spermatheca (SP; black dashed outline), sperm and embryos. gld-1(q485); gld-1^ndb::ollas worms accumulate oocytes, ovulate unfertilized oocytes and spermatheca appears empty. (C) Fluorescence micrograph of gld-1(q485); gld-1^wt::ollas or gld-1(q485); gld-1^ndb::ollas gonads dissected at L4 stage and immunostained for sperm (green) using anti-MSP antibody. A subset (8%) of gonads from gld-1(q485); gld-1^ndb::ollas worms lack sperm and exhibit feminized germline phenotype (Table 2). (D) Dissected gonads from either gld-1(q485); gld-1^wt::ollas or sterile gld-1(q485); gld-1^ndb::ollas worms stained with DAPI to reveal DNA morphology. Gonads from gld-1(q485); gld-1^ndb::ollas mutant worms exhibit endomitotic oocyte phenotype (white arrow) (27%; Table 2). Bar, 10 μm.

Figure 10

GLD-1 requires interaction with DLC-1 to prevent ectopic germline proliferation in vivo. Gonads were immunostained for mitotically dividing cells (pink), stem and progenitor cells (green), and DNA (blue) as in Figure 2. Dissected germlines from gld-1(q485); gld-1^wt::ollas transgenic animals treated with either (A) empty vector control or (B) gld-2(RNAi). Dissected gonads from gld-1(q485); gld-1^ndb::ollas mutant worms treated with either (C) control or (D) gld-2(RNAi). Efficacy of gld-2(RNAi) was determined by scoring sterility and embryonic lethality. gld-1(q485); gld-1^wt::ollas transgenic animals fed gld-2(RNAi) exhibited 100% sterility and gld-1(q485); gld-1^ndb::ollas mutant worms exhibited on average 86% sterility and the embryonic lethality of worms with eggs ranged from 43 to 100%. White arrow indicates aberrant cell proliferation. Images represent data collected from two independent experiments and 19–132 worms were scored for each genotype (see Table 3). Bar, 10 μm. (E) Mitotic region length was measured by counting REC-8 positive stem and progenitor cell row number of gld-1(q485); gld-1^wt::ollas (blue) and gld-1(q485); gld-1^ndb::ollas (green) transgenic animals fed either control, gld-2(RNAi), or gld-3(RNAi), respectively. A total of 22–80 germlines were scored for each genotype (N, indicated below the chart). A statistically significant difference in distribution of mitotic zone lengths was observed between gld-1(q485); gld-1^wt::ollas and gld-1(q485); gld-1^ndb::ollas worms treated with gld-3(RNAi) (indicated by *; P = 0.024) but not gld-1(q485); gld-1^wt::ollas vs. gld-1(q485); gld-1^ndb::ollas worms treated with gld-2(RNAi) (P > 0.4). Kolmogorov–Smirnov test was used to calculate P-values. Embryonic lethality of gld-1(q485); gld-1^wt::ollas; gld-3(RNAi) worms was 69 ± 25% and gld-1(q485); gld-1^ndb::ollas; gld-3(RNAi) was 54 ± 11%.

Figure 11

GLD-1 is a key regulator of germline development that affects multiple aspects of germ cells function. DLC-1 facilitates a subset of GLD-1 functions.