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First published online 4 October 2006
doi: 10.1242/dev.02614

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Pax2/5/8 proteins promote cell survival in C. elegans

Department of Molecular Genetics, Ohio State University, Columbus, OH 43210 USA

View larger version (104K): [in a new window]   Fig. 1. C. elegans Pax2/5/8 mutants have increased germline cell apoptosis. (A-H) Nomarski DIC (A,C,E,G) and epifluorescence (B,D,F,H) images of the gonad of adult hermaphrodite animals, stained with SYTO12, which preferentially labels dying germ cell nuclei (Gumienny et al., 1999). Arrowheads indicate dying cells or clusters of dying cells. (A,B) Wild type. (C,D) egl-38(n578). (E,F) pax-2(ok935). (G,H) pax-2(ok935) egl-38(n578). (I) Average number of cell corpses per gonad arm for wild type and Pax2/5/8 mutants. Error bars represent s.e.m. for at least 20 animals in each category. (J) Average number of cell corpses per gonad arm for different egl-38 mutants. Data presented as in I.

View larger version (21K): [in a new window]   Fig. 2. Induced expression of C. elegans Pax2/5/8 genes protects germline cells from apoptosis. (A) Induced expression of either egl-38 or pax-2 reduces germline apoptosis. Data presented as in Fig. 1. (B) Western blot for heat-treated lines in A, showing expression of the FLAG-tagged proteins. (C) Germline apoptosis assay in an engulfment-defective mutant background (ced-6) to increase the baseline number of cell corpses. Data presented as in Fig. 1.

View larger version (69K): [in a new window]   Fig. 3. C. elegans Pax2/5/8 mutants have increased somatic cell apoptosis. (A-D) Representative comma-stage embryos from different genotypes. Cell corpses visible in the plane of focus are indicated with an arrowhead. (A) Wild type. (B) egl-38(n578). (C) pax-2(ok935). (D) pax-2(ok935) egl-38(n578). (E) Average number of visible cell corpses in comma to 1.5-fold stage embryos for wild type and Pax2/5/8 mutants. lin-2(e1309) is included as a control for eggs retained in an egg-laying defective mother. Error bars represent s.e.m. for at least 45 animals in each category. (F) unc-4::gfp-positive VC neurons in wild type and Pax2/5/8 L2 stage mutant animals. (G) Genetic epistasis experiments between C. elegans Pax2/5/8 genes and other genes that affect somatic cell apoptosis. Animals of different genotypes were treated with RNAi induced by bacterial feeding (see Materials and methods) and assessed as in E.

View larger version (20K): [in a new window]   Fig. 4. Regulatory pathways that influence germline cell death. (A) The pathways that influence C. elegans germline cell apoptosis. Proteins discussed in the text are included. A physiological germ cell death is mediated through the ras pathway, with MPK-1 (MAP kinase) representing the most downstream component. Genotoxic treatments such as radiation induce checkpoint genes and p53-mediated apoptosis. Both pathways act to regulate a core pathway required for all normal cell deaths in C. elegans. EGL-38 and PAX-2 are drawn to act in parallel to the other two pathways, but could alternatively act downstream of MPK-1 (or CEP-1). (B) Genetic epistasis experiments between C. elegans Pax2/5/8 genes and other genes that affect germline apoptosis, carried out in an otherwise wild-type genetic background. (C) Genetic epistasis experiments between C. elegans Pax2/5/8 genes and other genes that affect germline apoptosis, carried out in animals RNAi-depleted for ced-10, a gene required for corpse engulfment. Germ-cell corpses were quantified as described in Materials and methods. Data are mean number of cell corpses per gonad arm±s.e.m. Wild type is N2.

View larger version (17K): [in a new window]   Fig. 5. Mosaic analysis for egl-38 function. (A) Cell lineage chart of the early cell divisions in C. elegans, indicating the embryonic source of the cells listed below, which were assayed in mosaic animals (see Sulston et al., 1983). An `x' indicates that the cells derive from more than one cell in the lineage. (B) Summary of the observations from mosaic animals, grouped according to category. For clarity, the mosaic animals were classified as wild type if they exhibited five or fewer corpses per gonad arm, and classified as mutant if they exhibited six or more corpses per gonad arm (beyond two standard deviations from the average of animals with the transgene present in all lineages). See text for additional analysis. All animals lacking the transgene in their offspring were classified as P4-negative. A more stringent analysis of germline loss (including in the analysis only animals that lack the transgene in D as well as P4 but retain the transgene in some other somatic lineages) yields a slightly lower, but similar proportion of animals classified as wild type (11 of 18, or 61%).

View larger version (24K): [in a new window]   Fig. 6. ced-9 acts downstream of the Pax2/5/8 genes. (A) ced-9 and mev-1 transcript abundance is altered in response to Pax2/5/8 gene activity. Quantitative RT-PCR results for ced-9, mev-1, ced-3, ced-4 and egl-1 genes in different Pax2/5/8 genetic backgrounds, normalized to the act-2 gene. Wild type (N2) and dpy-20 [the genetic background for egl-38(n578)] serve as controls. ced-9 and mev-1 transcript abundance is decreased in egl-38 pax-2 double mutants, and increased in response to induced expression of egl-38 or pax-2 (hsp::egl-38 and hsp::pax-2). The transcript abundance of the other cell death pathway genes is unaltered. Separate experiments indicate that ced-9 and mev-1 abundance is not altered in response to heat-shock in wild-type animals (data not shown). Error bars indicate standard deviation. (B) Induced expression of ced-9 can bypass the cell death defect of pax-2 egl-38 double mutants. By contrast, induced expression of mev-1 alone, or in combination with ced-9, does not significantly impact the cell death effect. Induced expression of pax-2 in wild type and pax-2 egl-38 mutants is included as a control. Data presented as in Fig. 1.

View larger version (46K): [in a new window]   Fig. 7. A deletion analysis identifies an upstream regulatory element important for ced-9 transcription. (A) Clones tested for ced-9::gfp expression and ced-9 rescue. The sequences coding for GFP are inserted into a unique PstI site in the third exon of ced-9. Deletions 1-3 correspond to three deleted clones that center on a potential Pax response element, indicated with a black bar on the clones. In the mutant clone, this element is altered, indicated by `X'. The sequence for both the wild-type and the mutant element is shown below the diagram. (B-I) Expression of the full length ced-9::gfp clone in embryos [(B,F) prior to elongation, (C,G) at comma stage, (D,H) at 1.5-fold stage] and (E,I) in adult gonad. (B-E) DIC images. (F-I) The same animals under epifluorescence to visualize GFP. In I, the bend of the gonad arm is shown and the ced-9 expression is marked with a bracket. (J-S) Representative embryos bearing each of the transgenes summarized in A. (J-N) DIC images. (O-S) The same embryos under epifluorescence to visualize GFP. All embryos are at a stage similar to that shown in B and F.

View larger version (28K): [in a new window]   Fig. 8. EGL-38 and PAX-2 directly regulate ced-9 transcription in vivo. (A) Diagram of the mev-1 ced-9 genomic region, showing the position of primers used for chromatin immunoprecipitation. Black arrowheads correspond to primers for the upstream region (UP); white arrowheads correspond to primers for the coding region (CR). (B) PCR of DNA recovered from chromatin immunoprecipitation. Input lane corresponds to DNA recovered without immunoprecipitation. Two representative experiments are shown, one each for a strain expressing EGL-38 (hsp::egl-38::FLAG) and PAX-2 (hsp::pax-2::FLAG). The experimental proteins (EGL-38 and PAX-2) are tagged at the C-terminus with a FLAG peptide (Fig. 2B), and can be immunoprecipitated with an -FLAG antibody. PCR of DNA recovered from immunoprecipitation using -FLAG antibody, protein A/G beads (negative control) or -Histone (positive control) are shown for representative experiments using EGL-38 (left) and PAX-2 (right). In both cases, the Pax2/5/8 protein preferentially immunoprecipitates DNA in the upstream region.

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