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Organ-specific cell division abnormalities caused by mutation in a general cell cycle regulator in C. elegans

Ivana Kostic and Richard Roy*

Department of Biology, McGill University, Montreal, Quebec, Canada



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  		Fig. 1. cdc-25.1(gf) mutants have increased numbers of intestinal nuclei. (A) Wild-type adult animals expressing elt-2::GFP, which marks intestinal nuclei. (B) rr31 adult showing an increased number of elt-2::GFP expressing nuclei. Scale bar: 25 µm. Anterior is towards the left.

 


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  		Fig. 2. Lineage analysis of the E blastomere in rr31 and cki-1(RNAi) animals. (A) Lineage map of the wild-type intestinal cell divisions during embryogenesis. Schematic representation of a characteristic lineage map of the (B) rr31 mutant and (C) cki-1(RNAi) intestinal cell divisions during embryogenesis. The vertical distances represent approximate time of development.

 


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  		Fig. 3. cdc-25.1 mutant sequence. (A) cdc-25.1 mutant DNA sequence including the translation start to up to and including exon 2. Exon sequence is in blue, intron sequence in red and the mutated nucleotide in yellow. (B) cdc-25.1 mutant cDNA sequence including exons 1 and 2. (C) CDC-25.1 mutant amino acid sequence (N-terminal region). The yellow amino acid indicates a G to D substitution at amino acid 47. The residues marked with red dots indicate a higher probability of being phosphorylated in the mutant while those marked with blue, indicate a decreased probability of being phosphorylated (PredictProtein software).

 


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  		Fig. 4. The cdc-25.1(gf) defect is specific to the E lineage. (A) mom-2 embryos produce extra mesoderm at the expense of endoderm, as seen by anti-PHA-4 staining, which marks pharyngeal precursors (descendants of the MS blastomere). (B) cdc-25.1(gf); mom-2 embryos have similar amounts of mesoderm as mom-2 mutants alone. (C,D) The embryos in A,B have similar cell numbers measured by counting DAPI stained nuclei: (C) mom-2 embryo in A; (D) the cdc-25.1(gf); mom-2 embryos in B. C. elegans embryos are approximately 50 µm in length.

 


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  		Fig. 5. cdc-25.1(gf) enhances the pop-1 phenotype. (A) Wild-type number of intestinal nuclei in 300 minute embryo visualized with elt-2::GFP. (B) cdc-25.1(gf) embryo at 300 minutes showing extra intestinal nuclei. (C) pop-1(RNAi) embryos have extra intestinal nuclei due to a MS to E transformation. (D) cdc-25.1(gf); pop-1(RNAi) embryos have twice as many intestinal nuclei as pop-1(RNAi) embryos alone. (E) Laser-mediated cell ablation of the E blastomere in cdc-25.1(gf) animals results in embryos arrested without any intestine as seen by the absence of elt-2::GFP expression. The embryo to the right is an unablated cdc-25.1(gf) mutant embryo, allowed to develop to late embryogenesis.

 


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  		Fig. 6. Heat-shock ectopic expression of cdc-25.1 causes entry into S phase in the intestinal cells. Posterior intestinal cells of adult hermaphrodites expressing the S-phase reporter (A) cye-1::GFP or (B) rnr::GFP after heat shock-induced expression of mutant cdc-25.1. (C) Posterior intestinal cells of adult hermaphrodites after heat shock. In C, animals harbor the S-phase reporter transgene cye-1::GFP and the empty heat-shock vector. Arrows indicate intestinal nuclei expressing the S-phase reporters. Scale bar: 10 µm. Anterior is leftwards.

 


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  		Fig. 7. The CDC-25.1(gf) protein perdures after the 28-cell stage. (A) Wild-type embryo at the 64-cell stage stained with anti-CDC-25.1 antibody (top) or DAPI (bottom), showing no apparent CDC-25.1 staining. (B) cdc-25.1(gf) embryo at the 100-cell stage stained with anti-CDC-25.1 antibody (top) or DAPI (bottom). (C) Enlarged cdc-25.1(gf) embryo stained with anti-CDC-25.1 showing nuclear staining as indicated by arrows (B,C). (D) The proportion of wild-type (hatched bar) or cdc-25.1(gf) (black bar) embryos that stain with anti-CDC-25.1 antibody up to the 28-cell stage, and after the 40 cell stage. n=48 and n=54 for wild type and cdc-25.1(gf), respectively.

 




© The Company of Biologists Ltd 2002