A functional genomic screen for evolutionarily conserved genes required for lifespan and immunity in germline-deficient C. elegans

Abstract

The reproductive system regulates lifespan in insects, nematodes and vertebrates. In Caenorhabditis elegans removal of germline increases lifespan by 60% which is dependent upon insulin signaling, nuclear hormone signaling, autophagy and fat metabolism and their microRNA-regulators. Germline-deficient C. elegans are also more resistant to various bacterial pathogens but the underlying molecular mechanisms are largely unknown. Firstly, we demonstrate that previously identified genes that regulate the extended lifespan of germline-deficient C. elegans (daf-2, daf-16, daf-12, tcer-1, mir-7.1 and nhr-80) are also essential for resistance to the pathogenic bacterium Xenorhabdus nematophila. We then use a novel unbiased approach combining laser cell ablation, whole genome microarrays, RNAi screening and exposure to X. nematophila to generate a comprehensive genome-wide catalog of genes potentially required for increased lifespan and innate immunity in germline-deficient C. elegans. We find 3,440 genes to be upregulated in C. elegans germline-deficient animals in a gonad dependent manner, which are significantly enriched for genes involved in insulin signaling, fatty acid desaturation, translation elongation and proteasome complex function. Using RNAi against a subset of 150 candidate genes selected from the microarray results, we show that the upregulated genes such as transcription factor DAF-16/FOXO, the PTEN homolog lipid phosphatase DAF-18 and several components of the proteasome complex (rpn-6.1, rpn-7, rpn-9, rpn-10, rpt-6, pbs-3 and pbs-6) are essential for both lifespan and immunity of germline deficient animals. We also identify a novel role for genes including par-5 and T12G3.6 in both lifespan-extension and increased survival on X. nematophila. From an evolutionary perspective, most of the genes differentially expressed in germline deficient C. elegans also show a conserved expression pattern in germline deficient Pristionchus pacificus, a nematode species that diverged from C. elegans 250-400 MYA.

Figure 1. Schematic of germline precursor cells, and cell ablation and microarray experiments.

In the C. elegans L1 stage larvae, the gonad and germline consist of four cells, namely Z1, Z2, Z3 and Z4 with Z(1,4) producing the gonad and Z(2,3) the germline. If Z(2,3) are killed with a laser beam then only somatic gonad tissue is left, and the animals are long-lived. However if Z(1,4) are ablated then the gonad and the germline are both killed as the germline cannot grow without the influence of the gonad and the lifespan of these animals is comparable to un-ablated controls. The different sets of microarray comparisons as described in the text are also indicated.

Figure 2. Effect of germline and gonad ablation on the survival of C. elegans exposed to pathogenic X. nematophila.

(A) Survival of C. elegans wild-type un-ablated (red) n = 50 (5 independent replicates), Z(2,3) germline-ablated (blue) n = 78 (8) and somatic gonad and germline ablation Z(1,4) (green) n = 48 (5) when exposed to X. nematophila. (B) Survival of germline-ablated Z(2,3) C. elegans wild-type (dark blue) n = 78 (8), Z(2,3)-ablated daf-16 (mu26) (turquoise) n = 35 (4), Z(2,3)-ablated daf-16 (mu27) (orange) n = 29 (3), Z(2,3)-ablated daf-16 (mu86) (light blue) n = 10 (1), Z(2,3)-ablated daf-12(m20) (pink) n = 14 (2), Z(2,3)-ablated tcer-1(tm1452) (purple) n = 15 (2), Z(2,3)-ablated mir-7.1(n4115) (green) n = 25 (3) and Z(2,3)-ablated nhr-80(tm1011) (red) n = 30 (3) when exposed to X. nematophila. (C) Survival of Z(2,3)-ablated C. elegans wild-type (blue) n = 78 (8) and Z(2,3)-ablated daf-2(e1370) mutants (red) n = 47 (5) when exposed to X. nematophila.

Figure 3. Summary of microarray experiments.

(A) Genes up-regulated in the Z(2,3) ablated vs. Z(1,4) ablated comparisons are significantly enriched for genes that are known to shorten lifespan upon knock-down by RNAi. (B) Significant enrichment of DAF-16 targets is observed in the genes up-regulated in Z(2,3)-ablated vs. Z(1,4)-ablated comparison.

Figure 4. RNAi screening for genes integral for survival towards X. nematophila from genome wide analysis.

(A) The effect of insulin signaling genes identified as being integral for survival towards X. nematophila from genome wide analysis. Survival of C. elegans glp-1(e2141); empty vector control (blue) n = 100 (2 independent replicates), glp-1(e2141); daf-16 (RNAi) (red) n = 100 (2) and glp-1(e2141); daf-18 (RNAi) (green) n = 100 (2) when exposed to X. nematophila. (B) The effect of proteasomal complex genes identified as being integral for survival towards X. nematophila from genome wide analysis. Survival of C. elegans glp-1(e2141); empty vector control (blue) n = 100 (2), glp-1(e2141); rpn-6.1 (RNAi) (red) n = 100 (2), glp-1(e2141); rpn-7 (RNAi) (green) n = 60 (2), glp-1(e2141); pbs-6 (RNAi) (purple) n = 100 (2), glp-1(e2141); rpn-10 (RNAi) (turquoise) n = 100 (2), glp-1(e2141); rpt-6 (RNAi) (orange) n = 100 (2) and glp-1(e2141); pbs-4 (RNAi) (light blue) n = 100 (2) when exposed to X. nematophila. (C) The effect of new genes identified as being integral for survival towards X. nematophila from genome wide analysis. Survival of C. elegans glp-1(e2141); empty vector control (blue) n = 100 (2), glp-1(e2141); T12G3.6 (RNAi) (purple) n = 100 (2), and glp-1(e2141); par-5 (RNAi) (orange) n = 60 (2) when exposed to X. nematophila.

Figure 5. Evolutionary conservation of genes differentially regulated upon germline ablation in C. elegans compared to P. pacificus.

(A) Significant overlap between germline-regulated genes in C. elegans and P. pacificus . Fisher's exact test p-value  = 3.04E-49. (B) Most of the germline regulated genes in C. elegans and P. pacificus also show similar expression patterns (Pearson correlation coefficient  = 0.7).