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lin-35 Rb and cki-1 Cip/Kip cooperate in developmental regulation of G1 progression in C. elegans

Massachusetts General Hospital Cancer Center, Building 149, 13th Street, Charlestown, MA 02129, USA

View larger version (49K): [in a new window]   Fig. 1. Identification and characterization of cyd-1 and cdk-4 mutant animals. (A) Positions of cells in the ventral cord precursor (P, green) and intestinal (I, gray) lineages in late L1 wild-type (top) and cell-cycle mutant (bottom) larvae. The lineages of an individual P and I cell are indicated for each genotype (right). (B) Postembryonic blast cells remain undivided in cyd-1 and cdk-4 mutants, as indicated for intestinal and P precursor cells in the enlarged sections (right). The panels show a late L1 wild-type larva (top) and similar stage cyd-1(he112) mutant (bottom) after fixation and DNA staining with propidium iodide. (C) Expression of the rnr::GFP S-phase marker in wild-type animal (left) and cyd-1(he112) mutant (right). Nomarski images (top) and corresponding epifluorescent images (bottom) show several cells of the P and intestinal lineages. In the cyd-1 animal, only autofluorescence of the intestinal cells is detectable. (D) Quantitative measurements of DNA content in the intestinal nuclei (gray bars) of wild-type animals and mutant strains of indicated genotype. Body wall muscle cells (black bars) serve as 2n DNA standards. Scale bars: 10 µm. Values indicated are mean±s.e.m.

View larger version (10K): [in a new window]   Fig. 2. Molecular lesions in cyd-1 and cdk-4 mutant alleles, illustrated with respect to the genomic structure. Exons are shown as boxes, introns as lines.

View larger version (15K): [in a new window]   Fig. 3. cyd-1 and cdk-4 cell division defects precede growth defects. Size of cyd-1(he112) and cdk-4(gv3) homozygous mutants and wild-type siblings (genotypes: cyd-1(he112)/mnC1 and cdk-4/+ or +/+) is plotted as a function of time of postembryonic development at 15°C. The growth retardation of cyd-1 and cdk-4 mutants becomes first apparent in the L2 stage, subsequent to failure of some late embryonic and all postembryonic L1 divisions. Points indicate mean of five measured animals±s.d.

View larger version (64K): [in a new window]   Fig. 4. lin-35 acts as a negative regulator of cell-cycle progression. (A) Expression of the rnr::GFP S-phase marker in cyd-1(he112) (left) and lin-35(RNAi); cyd-1(he112) (right). Nomarski images (top) and corresponding epifluorescent images (bottom) show several cells of the P and intestinal lineages. In the cyd-1 animal, only autofluorescence of the intestinal cells is detectable, whereas lin-35 RNAi resulted in expression of rnr::GFP in the P cells and other lineages. (B) Quantitative measurements of DNA content in the intestinal nuclei (gray bars) of wild-type (WT) and mutant animals of indicated genotype. Body wall muscles (black bars) serve as 2n DNA standard. (C) Rescue of postembryonic cell divisions by lin-35. The cell number in the P2-P10 and intestinal lineages were counted in animals of indicated genetic backgrounds. Scale bars: 10 µm. Values indicated are mean±s.e.m.

View larger version (17K): [in a new window]   Fig. 5. lin-35 is not rate limiting for S-phase entry. DNA content of intestinal cells of wild-type, lin-35(n745) and cki-1(RNAi) animals was determined at 1 hour intervals from the start of postembryonic development. Bars indicate mean of 10 intestinal nuclei±s.d.

View larger version (14K): [in a new window]   Fig. 6. cki-1 and cki-2 cooperate with lin-35 Rb in regulating G1 progression. (A) Quantitative measurements of DNA content in the intestinal nuclei (gray bars) of wild-type and mutant animals of indicated genotype. Body wall muscles (black bars) serve as 2n DNA standard. (B) Rescue of postembryonic cell divisions. The cell numbers in the P2-P10 and intestinal lineages were counted in strains of indicated genotype. Genes that do not carry allele designations were inhibited by RNAi. (C) Additional cell divisions in a wild-type background caused by inactivation of the indicated genes. The wild-type is defined as zero supernumerary divisions. In all three panels, bars indicate mean±s.e.m.

View larger version (29K): [in a new window]   Fig. 7. Cip/Kip and Rb family members cooperate in regulating G1 progression. Epifluorescent images demonstrating the number of intestinal divisions in (A) cyd-1(he112); elt-2::GFP mutant larvae and animals of the same genotype after (B) lin-35 RNAi, (C) cki-1,2 RNAi and (D) lin-35; cki-1,2 double RNAi. Intestinal nuclei express the GFP under the control of the elt-2 promoter region. Scale bars: 25 µm.