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First published online 28 February 2007
doi: 10.1242/dev.02818

Development 134, 1357-1368 (2007)
Published by The Company of Biologists 2007
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Timing of the onset of a developmental cell death is controlled by transcriptional induction of the C. elegans ced-3 caspase-encoding gene

Carine W. Maurer, Michael Chiorazzi and Shai Shaham*

Laboratory of Developmental Genetics, The Rockefeller University, 1230 York Avenue, New York, NY 10021, USA.


Figure 1
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  		Fig. 1. Expression of ced-3 promoter::GFP transgenes is induced shortly before tail-spike cell death. (A) Cartoon showing the tail-spike cell at different stages of development. Filled green ovals, tail-spike cell nuclei; gray oval, dying tail-spike cell corpse. Ball-stage embryo (left); threefold-stage embryo (center and right). (B-D) Merged DIC and GFP images of threefold-stage embryos expressing a 1.5 kb C. elegans ced-3 promoter::GFP reporter transgene (nsEx723). (B) Embryo before the onset of tail-spike cell GFP expression (arrow and inset: binucleate tail-spike cell). (C) Embryo at onset of tail-spike cell GFP expression (arrow and inset: bi-nucleate tail-spike cell). (D) Embryo approximately 31 minutes after the onset of tail-spike cell GFP expression (arrow and inset: dying tail-spike cell corpse). (E) Merged DIC and GFP image of a threefold-stage embryo expressing an egl-1 promoter::GFP integrated transgene (bcIs1). Arrow and inset: tail-spike cell corpse lacking GFP. Scale bars: 10 µm.

 

Figure 2
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  		Fig. 2. ced-3 is transcribed in many cells during developmental periods of cell death. Top and bottom panels for each image are DIC+fluorescence and fluorescence-alone images, respectively. (A) Image of a smg-1(r861) embryo expressing a transgene consisting of the full rescuing (7.6 kb) ced-3 genomic locus, into which GFP with a nuclear localization signal near the start codon was inserted (see Materials and methods). (B) Same as A. Notice cell corpse (circled) weakly expressing GFP. (C) A smg-1(r861) larva containing the same transgene as in A. Notice the absence of GFP expression. The observed fluorescence is caused by autofluorescence from gut granules. (D) A smg-1(r861) L4 male larva containing the same transgene as in A. Notice the bright expression in several cells in the tail. None of these cells is fated to die. (E) A smg-1(r861); ced-3(n717) larva containing the same transgene as in A. Notice the GFP expression in the tail-spike cell (arrows).

 

Figure 3
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  		Fig. 3. Three ced-3 promoter elements act redundantly and specifically to regulate cell death and ced-3 expression in the tail-spike cell. (A) Alignment of the A, B and C promoter regions from the nematodes C. elegans and C. briggsae. Conserved nucleotides are shaded; boxed nucleotides are deleted in B and C; consensus nucleotides of caudal/Cdx2-binding sites are indicated by asterisks above the relevant nucleotides; numbers indicate the positions relative to the ced-3 start codon. (B,C) Regions A, B and C are required for GFP reporter expression and for the rescue of the ced-3(n717) cell death defect in the tail-spike cell. All experiments were performed in ced-3(n717) mutants. Regions A, B or C were deleted in the context of the conserved 349 bp C. elegans ced-3 promoter (B), the 1.5 kb C. elegans ced-3 promoter (C, left data column), or of pJ40, a plasmid containing C. elegans ced-3 genomic DNA (C, middle and right data columns). Reporter expression and cell death rescue were assessed as described in the Materials and methods section. x, deleted region; % Tail-spike cells expressing GFP, average±s.e.m. (number of transgenic lines examined); % Tail-spike cells surviving, average±s.e.m. (2-3 transgenic lines); No. extra cells in anterior pharynx, average±s.d. (2-3 transgenic lines); N.A., not applicable.

 

Figure 4
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  		Fig. 4. pal-1 regulates ced-3 expression and cell death in the tail-spike cell. (A) A 0.7 kb C. briggsae ced-3 promoter::GFP reporter (nsIs25) is expressed in a surviving tail-spike cell (arrow; inset: GFP only) of a ced-3(n717) L2 larva. (B) Same reporter as in A in a pal-1(ns114); ced-3(n717) L2 larva imaged using the same exposure time (arrow: binucleate tail-spike cell; inset: GFP only). Notice the decreased GFP fluorescence in the tail-spike cell of the pal-1 mutant. (C) Image of a distinctive tail-spike cell corpse (green and inset) persisting in a ced-5(n1812); nsIs25 L1 larva. Anterior is to the left. (D) pal-1 is required for tail-spike cell death and acts in parallel to egl-1. For each genotype, between 20-52 L1 animals were scored for tail-spike cell corpses, as identified by position, shape and expression of the nsIs25 C. briggsae ced-3 promoter::GFP transgene. Error, s.e.m. Alleles used: ced-5(n1812), pal-1(ns114, ns115), egl-1(n1084n3082). Scale bars: 10 µm.

 

Figure 5
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  		Fig. 5. ns114 and ns115 are alleles of pal-1. (A) Mapping and rescue of ns114. Top: ns114/CB4856 heterozygotes were allowed to self-fertilize, and homozygous ns114 progeny were tested for the presence of CB4856 single nucleotide polymorphisms (SNPs). SNP locations are indicated by cosmid names above the rectangle depicting chromosome III, as is the location of the cosmid containing pal-1. The number of chromosomes containing CB4856-specific SNPs out of the total examined is indicated under each SNP. Bottom: the number of rescued lines out of the total number of transgenic lines examined is indicated; injections were performed into pal-1(ns114 or ns115); ced-3(n717); nsIs25 mutants. Details of the rescue experiment are described in the text and in the Materials and methods section. (B) Gene structure of pal-1 and location of the ns114 and ns115 lesions. Boxes indicate exons, lines indicate introns, darkened boxed region is the homeodomain-encoding region. (C) Comparison of the wild-type and ns114 mutant site to the consensus C. elegans splice donor sequence. Boxed region highlights the mutation. (D) Alignment between PAL-1 and its Drosophila and human homologs, Caudal and Cdx1/Cdx2. Conserved residues are shaded; asterisks indicate residues comprising the homeodomain; residue mutated by the ns115 mutation is boxed.

 

Figure 6
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  		Fig. 6. PAL-1 homeodomain binds to ced-3 promoter sequences. Electrophoretic mobility shift assay (EMSA) showing binding of labeled DNA derived from sites B and C (A and B, respectively) of the conserved C. elegans ced-3 promoter. Cold competitor DNA was added at concentrations of 10x (lanes 2, 7), 100x (lanes 3, 8), 200x (lanes 4, 9), 500x (lanes 5, 10) and 1000x (lanes 6, 11), as indicated by triangles above the autoradiogram. Lane 12 shows no binding of GST to labeled B or C DNA; lane 13 shows absent/weak binding of PAL-1 HD to labeled mutant oligonucleotide. Positions of shifted PAL-1 homeodomain-DNA complex and unbound DNA are indicated with arrows. (C) EMSA showing that wild-type DNA derived from site C can compete with labeled DNA derived from site B more effectively than mutant site C DNA. Cold competitor DNA was added at concentrations of 10x (lanes 2, 5), 100x (lanes 3, 6) and 1000x (lanes 4, 7). Details of the assay can be found in the Materials and methods section.

 

Figure 7
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  		Fig. 7. Model for the regulation of tail-spike cell death. EGL-1 and CED-9 (gray) play minor roles in regulating tail-spike cell death, whereas PAL-1-mediated transcription of ced-3 is a key regulatory module.

 

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© The Company of Biologists Ltd 2007