mett-10(oz36), a tumorous mutant. (A) A fluorescence micrograph of a dissected, DAPI-stained wild-type adult C. elegans hermaphrodite germ line, with schematic above. In the distal region, proliferating germ cells (2) reside in close contact with the somatic distal tip cell (1). At the transition zone (3), germ cells enter meiosis and proceed through meiotic prophase (4) to give rise to sperm in the L4 stage (6) and to oocytes during adulthood (5). (B and C) Fluorescence micrographs of distal germ lines labeled for 3 hr with EdU from (B) wild type and (C) mett-10(oz36) animals. (D and E) Extended focus projections of distal germ lines from (D) wild type and (E) mett-10(oz36) animals stained for the M-phase marker pH 3. Yellow dotted lines indicate the approximate boundary of the proliferative and transition zones; white arrowheads indicate distal tip. All images from animals raised at 15°. Bar, 20 μm.
C. elegans METT-10 encodes a putative methyltransferase. (A) Alignment of the methyltransferase-10 domain of C. elegans METT-10 (GenBank accession no. NP_499247), Homo sapiens METT10D (GenBank accession no. NP_076991), and Xenopus laevis MGC81116 (GenBank accession no. NP_001085334). The architecture of the methyltransferase domain is shown both by identification of methyltransferase motifs (Maloneet al. 1995) and by secondary structure predictions with α-helices in pink and β-sheets in green (see materials and methods). Motifs I, II, III, IV, V, and X were identified by examination of the human METT10D N-terminal crystal structure (PDB 2H00), which did not include the region predicted to contain motifs VI and VIII (see materials and methods). Previous work suggests that Motifs X, I, and III are primarily responsible for binding to SAM, while Motifs IV, VI, and VIII are primarily responsible for enzyme catalysis (Maloneet al. 1995). Red arrows show the location of residues with significant contacts with SAM in the crystal structure of human METT10D, including G110 (large red arrow) in Motif I which is mutated to arginine in mett-10(oj32). G110 normally provides direct contact with the methionine group of SAM. The overall structure and ability to bind SAM appears highly conserved between orthologs, with the exception of an insertion of a predicted (indicated by an asterisk) α–helix between β4 and α5 in C. elegans. Not depicted is the C-terminal half of the METT-10 protein, which may contain the target recognition domain and is significantly less conserved between orthologs. (B) Domain architecture of METT-10 marked with protein sequence changes caused by mett-10 alleles. Shading highlights the conserved methyltransferase-10 domain.
Loss of mett-10 function causes multiple temperature-sensitive somatic defects. Vulva of (A) wild type, (B) mett-10(oz36), and (C) mett-10(ok2204) animals raised at 25°. mett-10 mutants have the protruding vulva phenotype (black arrow). (D) A normal posterior region of intestine in a wild-type animal. White arrowheadss mark the borders of the intestinal lumen. (E) mett-10(oz36) and (F) mett-10(ok2204) animals raised at 25° have distended intestinal lumens containing visible bacteria. (G and H) Phenotypes of progeny of smg-2(e2008); mett-10(oz36) animals at 20°. (G) Most embryos arrest during late embryonic development with visible vacuoles (black arrows), (H) embryos are often abnormal in size (see small embryo on left), and (I) the progeny that do hatch arrest shortly afterward with visible abnormalities (e.g., white arrow).
Germ-line phenotypes of mett-10(ok2204). (A) A 1-μm confocal slice of the distal end of a wild-type germ line stained with the meiotic marker GLD-1. (B) Confocal slices of representative distal germ lines from mett-10(ok2204) m-z- animals raised at 25° and stained with an antibody against GLD-1. Phenotypes range from small distal regions containing enlarged nuclei (white arrows) to gross morphological abnormalities with multiple gonadal protrusions (larger yellow arrows). Many of the protrusions stain with the meiotic marker GLD-1, suggesting that they are often filled with meiotic cells and do not represent duplications of the distal tip. These gonad abnormalities are not observed for any mett-10 allele at 15°. (C) A confocal slice of a wild type germ line labeled with EdU for 4 hr at 15° and stained with anti-phosphohistone-3 to mark cells in M-phase. With 4 hr of labeling, essentially all cells within the proliferative zone are labeled. (D) A mett-10(ok2204) germ line labeled with EdU for 4 hr at 15° and stained for phospho-histone-3. Large, diffuse nuclei are visible in the distal region (white arrows); these cells do not label with EdU in this time period and do not appear to be in M-phase (absent for pH 3). (E) Fluorescence micrograph of a wild-type proximal germ line stained with an antibody against lamin to visualize oocyte nuclear positions. Wild-type animals produce a single row of oocytes. (F) The proximal germ line of a mett-10(ok2204) animal shifted from 20° to 25° for 2 days as an L4. Disorganization of the oocytes and abnormal chromosomal morphology are visible. Bars, 20 μm.
mett-10(oz36) overproliferation is glp-1 dependent. Fluoresence micrographs of germ lines stained with DAPI to visualize DNA morphology (blue) and for the meiotic marker GLD-1 (green). (A and A′) In wild-type hermaphrodites, GLD-1 levels increase as germ cells enter meiosis and decrease as they transition from pachytene to diplotene (Joneset al. 1996). (B and B′) Homozygous mett-10(oz36) animals raised at 15° exhibit a late-onset tumorous phenotype, as evidenced by an increase in length of the distal proliferative zone and a spatial delay in GLD-1 accumulation. (C and C′) Disruption of nonsense-mediated mRNA decay with a mutation in smg-2 significantly enhances the penetrance (Table 1) and degree of tumor formation of mett-10(oz36) at 20°, resulting in germ lines containing proliferating cells throughout. (D and D′) At the permissive temperature of 20°, glp-1(bn18) hypomorphic temperature-sensitive mutants have only slight reductions in distal proliferative zone size. (E and E′) glp-1(bn18) fully suppresses the tumorous phenotype of smg-2(e2008); mett-10(oz36) animals at 20°. Yellow dotted lines indicate the approximate boundary of the proliferative and transition zones. Bar, 20 μm.
mett-10(g38) suppresses the temporal dynamics of premature meiotic entry in glp-1(bn18). (A–I) Extended focus projections of distal germ lines stained with DAPI to visualize DNA (blue) and for the M-phase marker pH 3 (pink). White dotted lines indicate the approximate boundary between the proliferative and transition zones as defined by DNA morphology. (A–D) Wild-type and mett-10(g38) animals have similar proliferative zone size and number of pH 3-positive nuclei at 20° and exhibit small decreases in the number of pH 3-positive nuclei after animals are shifted to 25° for 6 hours. (E and F) After a 6-hr shift to 25°, no pH 3-positive nuclei can be found in glp-1(bn18). (G and H) After a 6-hr shift to 25°, glp-1(bn18) mett-10(g38) germ lines still contain pH 3-positive nuclei and retain a longer proliferative zone than glp-1(bn18) alone. (I). After an 18-hr shift to 25°, no pH 3-positive nuclei can be found in glp-1(bn18) mett-10(g38) germ lines, and nuclear morphology is consistent with all cells entering meiosis with transition zone nuclei located immediately adjacent to the distal tip (white arrow). (J) Quantification of A–H. Bar, 20 μm.
METT-10∷GFP is a nuclear protein expressed in multiple cell types. (A–C) Imaging of METT-10∷GFP in live animals. (A) METT-10∷GFP can be seen in intestinal nuclei (intestine delineated by dashed line), (B) in the developing vulval epithelium (white arrows; seen here at L4 stage), spermatheca (yellow dotted lines), and (C) the adult vulva. (D–G) METT-10∷GFP expression in the somatic gonad and germ line. METT-10∷GFP is expressed in the nuclei of (D) the distal tip cell (white arrow), (E) sheath cells, (F) oocytes, and (G and G′′) pachytene germ cells, where localization appears largely distinct from DNA.
The pattern of METT-10∷GFP accumulation in the distal germ line is altered in glp-1 mutants. Confocal slices of 1 μm are shown. Proliferating germ cells were labeled with a 3-hr pulse of EdU (red). METT-10∷GFP (green) accumulates in nuclei as cells enter meiosis, although some faint staining is also observed in some proliferative nuclei. (B) A 6-hr shift of glp-1(bn18) animals to 25° results in almost all germ cells entering meiosis and in a concomitant shift in the pattern of METT-10 accumulation. (C) glp-1(ar202gf) animals shifted for a day to 25° at the L4 stage form germ-line tumors. METT-10 was observed in proliferating germ cells, although lower levels tended to be detected in nuclei that are labeled with EdU (regions encircled by white dotted lines). Bar, 20 μm.
mett-10 acts in the germ line to inhibit proliferative fate. To determine the focus of METT-10 action in proliferative fate decisions, mosaic analysis was carried out using an mett-10(+) extrachromosomal array marked with sur-5∷gfp [ozEx63] (Yochemet al. 1998). Animals were individually scored for presence of the array in the germ line (presence in progeny), cells of the somatic gonad (gonadal sheath and distal tip cell), and the intestine. Location of array loss within the lineage was inferred by the absence of GFP in all scored tissues descending from a given cell. Green circles indicate the presence of an array, while open circles indicate its absence. Loss of the rescuing array in the germ-line lineage results in a failure to suppress the fully penetrant tumorous phenotype of the smg-2 (e2008); mett-10(oz36) double mutant, while loss in the somatic gonad is tolerated. One animal had one tumorous germ line and one nontumorous germ line, both with GFP+ somatic gonads. We infer that this is likely a later loss in one of the germ-line precursors Z2 or Z3.
↵a Full genotypes of the mutant combinations are unc-32(e189) mett-10(oz36)/nDf40 dpy-18(e364), unc-32(e189) mett-10 (oz36)/mett-10(ok2204) dpy-18(e364), smg-2(e2008); unc-36(e251) mett-10(oz36)/unc-119 (ed3), smg-2(e2008); unc-36(e251) mett-10(oz36)/unc-32(e189) mett-10(g38). All animals were m+z-; i.e., homozygous mutants (z-) from mothers carrying a wild-type copy of mett-10.
↵b Animals grown at 15° were scored at 3 days past L4 and animals grown at 20° were scored at 2 days past L4.
↵c Tumorous includes late onset and proliferation throughout the germ line, although some germ cells in various meiotic stages may be observed. Phenotype scored by DIC.
Loss of mett-10 function leads to multiple, temperature-sensitive phenotypes rescued by maternal gene product
Pvl, protruding vulva; Ste, Sterile (no embryos present); Mel, maternal-effect lethal (lays dead embryos); NA, not applicable, by definition, since sterile mutants lay no embryos and thus cannot be Maternal-effect lethal.