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First published online 19 September 2007
doi: 10.1242/dev.004606

Development 134, 3691-3701 (2007)
Published by The Company of Biologists 2007
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C. elegans orthologs of components of the RB tumor suppressor complex have distinct pro-apoptotic functions

Claus Schertel and Barbara Conradt*

Dartmouth Medical School, Department of Genetics, Norris Cotton Cancer Center, 7400 Remsen, Hanover, NH 03755, USA.


Figure 1
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  		Fig. 1. The loss of lin-35, dpl-1, efl-1 or efl-2 function results in a reduced level of constitutive germ cell apoptosis. (A) Time-course analysis of constitutive germ cell apoptosis, which was analyzed in C. elegans hermaphrodites of the indicated genotypes. Average numbers of apoptotic germ cells per gonad arm of four independent experiments are shown. Error bars represent standard deviations. For each experiment, a minimum of ten animals per genotype and time point was scored double blind. All strains analyzed were homozygous for the mutation ced-6(n2095). Except for the strain efl-1(n3318), all strains were also homozygous for the Plim-7ced-1::gfp integration bcIs39. m+z- indicates that animals analyzed were homozygous mutant progeny of heterozygous animals. The complete genotypes of dpl-1(n3316) and efl-1(n3318) animals were dpl-1(n3316) unc-4(e120); ced-6(n2095); bcIs39 and ced-6(n2095); efl-1(n3318), respectively. (B) Analysis of constitutive germ cell apoptosis 40 hours post the L4 stage. Average numbers of apoptotic germ cells per gonad arm of two independent experiments are shown. Error bars represent standard deviations. For each experiment, a minimum of ten animals per genotype was scored blind. All strains were homozygous for ced-6(n2095) and bcIs39. Mutations of lin-35, dpl-1 and efl-2 used were: lin-35(n745), dpl-1(n3643) and efl-2(tm2359).

 

Figure 2
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  		Fig. 2. Role of ced-9 in lin-35-dependent germ cell apoptosis. (A) The loss of lin-35 function suppresses germ cell apoptosis caused by the partial loss of ced-9 function. DIC and fluorescence images of gonad arms of ced-9(n1653ts) and lin-35(n745); ced-9(n1653ts) C. elegans hermaphrodites. Both strains were homozygous for the Plim-7ced-1::gfp integration bcIs39. White arrows point to apoptotic germ cells. (Aa) Gonad of a ced-9(n1653ts) hermaphrodite that is severely damaged. (Ab) Gonad of a ced-9(n1653ts) hermaphrodite that is not severely damaged but contains a large number of apoptotic germ cells. (Ac,Ad) Gonads of lin-35(n745); ced-9(n1653ts) hermaphrodites that are not severely damaged and contain low numbers of apoptotic germ cells. (B) The dosage of ced-9 determines the level of constitutive germ cell apoptosis. Constitutive germ cell apoptosis was analyzed 40 hours post the L4 stage. Average numbers of apoptotic germ cells per gonad arm are shown. Error bars represent standard deviations of three independent experiments. For each genotype, a minimum of 20 animals was scored blind (n=20-51). The complete genotype of the animals was: ced-1(e1735) [control (+/+)], ced-1(e1735); nDf20; nDp2[unc-86(e1416)] (+/-), ced-1(e1735); nDf20/dpy-17(e164); nDp2[unc-86(e1416)] (+/+) and ced-1(e1735); dpy-17(e164); nDp2[unc-86(e1416)] (+/+/+).

 

Figure 3
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  		Fig. 3. Analysis of ced-9, ced-4, and ced-3 expression in lin-35 and dpl-1 mutant gonads. (A) Quantitative real-time RT-PCR experiments using cDNAs isolated from gonads dissected from wild-type (+/+), lin-35(n745) or dpl-1(n3643) C. elegans. Average relative abundances of ced-9, ced-4 and ced-3 mRNAs of three independent experiments, each performed in triplicate, are shown. Error bars represent standard deviations. (B) Western analyses using proteins extracted from gonads dissected from wild-type (+/+), lin-35(n745) and dpl-1(n3643) hermaphrodites. The protein ß-actin [lin-35(n745)] or ß-tubulin [dpl-1(n3643)] was used as loading control. One representative experiment is shown for each. (C) Quantification of the abundance of CED-9 and CED-4 proteins in the gonad of wild-type (+/+), lin-35(n745) and dpl-1(n3643) animals. Quantification was performed using NIH image software. Average relative abundances of CED-9 and CED-4 protein of three [lin-35(n745) and two dpl-1(n3643)] independent experiments are shown. Error bars represent standard deviation.

 

Figure 4
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  		Fig. 4. Response to DNA damage in lin-35, dpl-1, efl-1 and efl-2 mutants. (A) Analysis of DNA-damage-induced germ cell apoptosis. For the data represented by the black bars, hermaphrodites were synchronized and irradiated at the L4 stage and germ cell apoptosis analyzed 24 hours post-irradiation. For the data represented by the gray bars, hermaphrodites were synchronized at the L4 stage, irradiated 12 hours post the L4 stage, and germ cell apoptosis analyzed 24 hours post-irradiation. Average numbers of apoptotic germ cells per gonad arm of four (black bars; n=61-79) or three (gray bars; n=38-54) independent experiments are shown. Error bars represent standard deviations. Strains were scored blind. m+z- indicates that animals analyzed were homozygous mutant progeny of heterozygous animals. (B) Semi-quantitative egl-1 RT-PCR experiments using cDNAs isolated from unirradiated (0 Gy) or irradiated (100 Gy) wild-type (+/+), lin-35(n745) [lin-35(lf)], dpl-1(n3643) or efl-2(tm2359) animals. Purified cDNA was used as positive control (+), water as negative control (-). tbg-1 RT-PCR was performed as the control. Representative experiments of three [lin-35(n745)] and two [dpl-1(n3643), efl-2(tm2359)] independent experiments are shown. (C) Quantitative egl-1 RT-PCR experiments using cDNAs isolated from unirradiated (0 Gy) or irradiated (100 Gy) wild-type (+/+) or lin-35(n745) gonads. Average relative mRNA abundances of two or three independent experiments, each performed in triplicates, are shown. Error bars represent standard deviations.

 

Figure 5
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  		Fig. 5. Genetic pathways for germ cell apoptosis in C. elegans. (A) The genetic pathway for constitutive germ cell apoptosis. The genes lin-35, dpl-1, efl-2 and, most likely, efl-1, promote constitutive germ cell apoptosis by blocking the function of the anti-apoptotic gene ced-9 or by enhancing the function of the pro-apoptotic genes ced-4 and ced-3, respectively. Furthermore, a lin-35, dpl-1, efl-1 and efl-2-independent pathway most likely acts in parallel to lin-35, dpl-1, efl-1 and efl-2 to promote constitutive germ cell apoptosis. Solid lines represent confirmed genetic interactions. (B) The genetic pathway for DNA damage-induced germ cell apoptosis. lin-35, dpl-1 and efl-2 function downstream of or in parallel to cep-1 to cause DNA damage-induced germ cell apoptosis by controlling the expression of unknown target genes. See text for details.

 




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