Hemizygosity of mdf-1 causes a defect in starvation-induced cell-cycle arrest of PGCs. (A) Fluorescence micrographs of N2 (wild-type) and Δmdf-1/+ worms in L1 diapause are shown. Newly hatched worms were starved for 4 days and then fixed. DNA was stained with DAPI (blue), and germ cell-specific P granules were stained with anti-PGL-1 antibody (red). Germ cells (PGL-1⁺ cells) are indicated by white arrowheads. Scale bar, 15 μm. (B) Newly hatched worms of the indicated genotypes were starved for 4 days and then fixed. DNA and germ cell-specific P granules were stained with DAPI and anti-PGL-1 antibody, respectively. The number of PGL-1⁺ cells per worm was counted, and the distribution of the number of germ cells per worm is plotted. (C) The level of MDF-1 expression in Δmdf-1/+ larvae. Whole-worm lysates prepared from N2 or Δmdf-1/+ larvae were separated on a gel, transferred to a nitrocellulose membrane and then probed with antibodies against MDF-1 and α-tubulin. The amount of lysate loaded into each well corresponded to that prepared from the indicated number of adult gravid worms. (D) The time course of germ cell proliferation during L1 diapause. N2 (blue plot) and Δmdf-1/+ (red plot) larvae were hatched on NGM plates with no food, and a portion of each group of worms was then fixed every day for 4 days. Fixed worms were stained with DAPI and anti-PGL-1 antibody, and the germ cells were counted. The average number of germ cells per worm is plotted.

Inappropriate germ cell proliferation in Δmdf-1 hemizygotes and Δdaf-18 homozygotes. Newly hatched worms of the indicated genotypes were starved for 4 days and then fixed. DNA and germ cell-specific P granules were stained with DAPI and anti-PGL-1 antibody, respectively. The number of PGL-1⁺ cells per worm was counted, and the distribution of the number of germ cells per worm is plotted. Note that the plot of wild-type N2 data is a duplicate of the data shown in .

The fzy-1(h1983) hypomorphic allele of fzy-1/CDC20 partially suppressed that caused by the loss of DAF-18. Newly hatched worms of the indicated genotypes were starved for 4 days and then fixed. DNA was stained with DAPI, and germ cell-specific P granules were stained with anti-PGL-1 antibody. The number of PGL-1⁺ cells per worm was counted, and the distribution of the number of germ cells per worm is plotted. Note that the charts of Δmdf-1/+ and Δdaf-18 are duplicates of the data shown in and , respectively.

MDF-1 is phosphorylated at Thr523 and Thr672 in vitro in an AKT kinase-dependent manner. (A) Bacterially expressed GST protein and recombinant proteins consisting of fusions between GST and peptides derived from various regions of MDF-1 (the positions of the amino acids corresponding to each peptide are indicated) were purified with glutathione sepharose. Indicated proteins were incubated with Akt1 kinase and [γ-³²P]ATP at 30°C for 30 min, eluted with the SDS sample buffer, and separated on the gel. Incorporation of [γ-³²P]ATP was analysed by autoradiography. GSK-3 is a fusion protein containing the Akt1 phosphorylation motif of hGSK-3. (B) Indicated proteins, in the amount used in the Akt1 kinase reaction, were loaded, separated on the gel and then stained with Coomassie blue dye. (C) Schematic structure of MDF-1. The predicted MDF-2-binding domain is indicated by the dark grey bar. Consensus Akt kinase phosphorylation sites ([R/K]XX[T/S] or RXRXX[T/S]) present in peptides phosphorylated by Akt1 kinase in vitro are shown in bold letters. (D) Immunoprecipitations were performed using an affinity-purified anti-MDF-1 rabbit polyclonal antibody on extracts from L1 larvae of the indicated genotypes that had been starved for 4 days. The immunoprecipitants separated on a gel were transferred to the nitrocellulose membrane and then probed with phospho-Akt substrate-specific antibody (anti-RXRXXSp/Tp) or with anti-MDF-2 antibody. The probe was stripped, and then the membrane was reprobed with anti-MDF-1 antibody. (E) The yeast two-hybrid analysis was performed to test the binding activity of wild-type (MDF-1wt) or mutated MDF-1 proteins (MDF-1T523D, MDF-1T523A) to MDF-2. The yeast strain Y190 was transformed with the indicated combination of plasmid vectors, then the growth was analysed on agar plates of synthetic media that was −Leu, −Trp, (His+) or −Leu, −Trp, −His containing 20 mM 3-AT (His−+20 mM 3-AT). Wild-type or mutant MDF-1 was cloned into the pACT vector, and MDF-2 was cloned into the pGBT9 vector. Vector: empty vector.

MDF-1T523AT672A partially suppressed the inappropriate germ cell proliferation caused by the loss of DAF-18. (A) Immunoprecipitations were performed using an anti-GFP rabbit polyclonal antibody on extracts from Δdaf-18, Δmdf-1 L1 larvae expressing either GFP∷MDF-1(wild-type) or GFP∷MDF-1T523AT672A that were starved for 4 days. The immunoprecipitants separated on a gel were transferred to the nitrocellulose membrane and then probed with anti-MDF-1 antibody or phospho-Akt substrate-specific antibody (anti-RXRXXSp/Tp). (B) Newly hatched worms of the indicated genotypes were starved for 4 days and then fixed. DNA and germ cell-specific P granules were stained with DAPI and anti-PGL-1 antibody, respectively. The number of PGL-1⁺ cells per worm was counted, and the distribution of the number of germ cells per worm is plotted. The plots shown in black indicate the suppression of inappropriate germ cell proliferation.

Germline apoptosis and reduction of brood size in mdf-1 hemizygotes released from starvation are cep-1 dependent. (A) Hermaphrodites of the indicated strains were grown in the presence of OP50 bacteria or starved for 4 days and then fed. The number of fertilized eggs laid per worm was counted for three individual worms, and the proportion of the fertilized eggs laid by temporarily starved worms compared with that laid by worms grown under normal conditions is shown. (B) Fluorescent microscope images of gonads of CED-1∷GFP-expressing strains with indicated genetic backgrounds are shown. The worms were starved for 4 days and then fed for 2 days. Apoptotic cells surrounded by CED-1∷GFP are indicated by white arrows. Scale bar, 15 μm. (C) The indicated strains were either grown for 2 days after hatching (pale grey bars) or starved for 4 days and then fed for 2 days (dark grey bars). The mean number of apoptotic cells per gonad arm of the indicated strains is plotted. (D) The differential interference contrast (DIC) microscope images (left panels) and fluorescent microscope images (right panels) of gonads of strains with the indicated genotypes stained with SYTO12 are shown. The newly hatched L1 larvae were starved for 4 days and then fed for 2 days. Apoptotic cells were positively stained with SYTO12. Scale bar, 15 μm. (E) The average numbers of SYTO12⁺ cells per gonad arm in worms with the indicated genotypes are plotted.

Proposed model of the pathway that controls germ cell proliferation during L1 diapause. MDF-1 causes the starvation-induced cell-cycle arrest of PGCs by inhibiting the activity of the APC/C^CDC20. MDF-1 is a downstream target of AKT-1. Phosphorylation of MDF-1 by AKT-1 reduces MDF-1's ability to bind MDF-2. DAF-18 indirectly activates MDF-1 by negatively regulating the AGE-1–AKT-1 signalling pathway. MDF-1–FZY-1 may not be the sole downstream factor of this pathway; another as yet unknown AKT-1 target may also regulate germ cell proliferation.