A fln-2 mutation affects lethal pathology and lifespan in C. elegans

Abstract

Differences in genetic background in model organisms can have complex effects on phenotypes of interest. We previously reported a difference in hermaphrodite lifespan between two wild-type lines widely used by C. elegans researchers (N2 hermaphrodite and male stocks). Here, using pathology-based approaches and genome sequencing, we identify the cause of this difference as a nonsense mutation in the filamin gene fln-2 in the male stock, which reduces early mortality caused by pharyngeal infection. We show how fln-2 variation explains previous discrepancies involving effects of sir-2.1 (sirtuin deacetylase) on ageing, and show that in a fln-2(+) background, sir-2.1 over-expression causes an FUDR (DNA synthesis inhibitor)-dependent reduction in pharyngeal infection and increase in lifespan. In addition we show how fln-2 variation confounds effects on lifespan of daf-2 (insulin/IGF-1 signalling), daf-12 (steroid hormone signalling), and eat-2 (putative dietary restriction). These findings underscore the importance of identifying and controlling genetic background variation.

Fig. 1

N2M is resistant to pharyngeal infection. a N2M (dashed lines) is longer lived than N2H (solid lines) (+16.6%, p < 0.0001, log rank test), yet neither P or p N2M subpopulations are long lived. N2M longevity disappears after treatment with antibiotics. Whole population in black, P death sub-population in red, p death in blue, and antibiotic-treated in green. See Supplementary Table 1 for full statistics, and Supplementary data 1 for raw mortality data. b Necropsy analysis of the two N2 lines. N2M experience less P death than N2H. Error bars represent s.e.m. of six trials. p = 0.0031, Student’s t test. c Antibiotic and phm-2 mutants suppress P at different stages. Pharyngeal invasion and widespread infection were scored using high magnification epifluorescence microscopy in worms fed RFP-expressing bacteria. No detectable RFP within pharyngeal tissue is shown in white, initial invasion with RFP puncta in pink, widespread RFP in red, and worms that have died with swollen pharynxes in maroon. Mean of two trials, n = 18–20 per time point in each trial. d N2M is more resistant to initial bacterial invasion. Colour coding as in (c). Mean of two trials, n = 20–25 animals per time point in each trial. e Reduced P frequency in N2M is not a consequence of parental mating. Error bars represent s.d. of two biological replicates. p = 0.0234 for N2M × N2M mating progeny, Student’s t test. f Resistance to P death following successive rounds of backcrossing is consistent with Mendelian segregation of a single mutant locus. Error bars represent s.d. of two biological replicates. Asterisk indicates p < 0.05, Student’s t test. See Material and Methods for detail. Source data for (b–f) are provided as a Source Data file

Fig. 2

Resistance to P death is caused by a recessive mutation in fln-2. a Design of Variant Discovery Mapping strategy to select the recessive low P phenotype of N2M in F3 progeny. b Frequency of parental (N2M genotype) reads on chromosome X. For results for all chromosomes see Supplementary Fig. 3. c Normalised frequency of pure parental (N2M genotype) reads on chromosome X. d Illustration of the fln-2(ot611) mutation on FLN-2A, the longest isoform of fln-2. e Necropsy analysis and f lifespan of N2H-derived fln-2(syb202) Y800* strain. N2H survival curve in black, N2M in blue, and fln-2 Y800* in purple. Asterisks indicate p < 0.01, Student’s t test. Error bars represent s.e.m. of three trials. See Supplementary Table 2 for full statistics. See Supplementary data 1 for raw mortality data. Source data are provided as a Source Data file

Fig. 3

Reduction of P death by fln-2(ot611) masks effects of high copy number sir-2.1 over-expression (oe) on lifespan. a Lifespan and b necropsy analysis of sir-2.1(oe) and control strains used in previous studies. GA strains are indicated by solid lines: GA468 geIs3 sir-2.1(oe); fln-2(ot611) (blue) and GA707 wuEx166; fln-2(ot611) control (black). LG strains are indicated by dashed lines: LG394 geIs3 sir-2.1(oe) (blue) and LG398 geIs101 control (black). sir-2.1(oe) reduced P death and extended lifespan in LG strains but not GA strains, consistent with both previous, conflicting studies^,. Error bar represents s.e.m. of three trials. See Supplementary Table 4 for full statistics. See Supplementary data 1 for raw mortality data. Source data are provided as a Source Data file. c Lifespan and d necropsy analysis of sir-2.1(oe) and control strains after backcrossing four times with N2H. GA outcrossed in solid lines: GA1909 geIs3 sir-2.1(oe) (blue) and GA1907 wuEx166 control (black); LG outcrossed in dashed lines: GA1913 geIs3 sir-2.1(oe) (blue) and GA1915 geIs101 control (black). sir-2.1(oe) strains (blue) from both labs showed a reduced frequency of P death compared to control strains (black) in fln-2(+) background. See Supplementary Table 5 for full statistics. a–d Trials conducted in the presence of 10 μM FUDR. See Supplementary data 1 for raw mortality data. e Lifespan and f necropsy analysis of backcrossed sir-2.1(oe) and control strains, with FUDR excluded. Colour coding as in (c, d). See Supplementary Table 7 for full statistics. See Supplementary data 1 for raw mortality data. g sir-2.1(oe) and 10 μM FUDR interact to protect against progression of bacteria infection. Pharyngeal invasion and widespread infection were scored in worms fed RFP-expressing bacteria. Colour coding as in Fig. 1c. Mean of two trials, n = 16–20 per time point in each trial

Fig. 4

Effect of daf-12 on daf-2 at 25 °C is partially affected by fln-2(ot611) variation. a Lifespan and b necropsy analysis of daf-2(m41); daf-12(m20) and daf-2(m41) mutants in fln-2(+) or fln-2(ot611) backgrounds. daf-2 fln-2 in green, daf-2 in blue, and daf-2 daf-12 in purple. Asterisks indicate significant differences, p < 0.0001 and 0.01, respectively. Error bars represent s.d. of two trials. See Supplementary Table 10 for full statistics, and Supplementary data 1 for raw mortality data. Source data are provided as a Source Data file. c Lack of lifespan effect by daf-12 in daf-2 p sub-population lifespan in fln-2(+) background. See Supplementary data 1 for raw mortality data

Fig. 5

Reduction of P death by fln-2(ot611) masks lifespan effects of eat-2. a Necropsy analysis and b lifespan of eat-2(ad1116) mutants in fln-2(+) or fln-2(ot611) backgrounds. N2H survival curve indicated by solid black line, N2M by dashed black line, eat-2 by solid purple line, and eat-2 fln-2 by dashed purple line. Error bars represent s.e.m. of three trials. See Supplementary Table 11 for full statistics, and Supplementary data 1 for raw mortality data. Source data are provided as a Source Data file. c eat-2(ad1116) causes a small but significant increase in p sub-population lifespan in both fln-2(+) and fln-2(ot611) backgrounds. See Supplementary data 1 for raw mortality data. d Effects of fln-2(ot611) on lifespan in eat-2(ad1116) mutants disappears after treatment with antibiotics (200 μg/ml carbenicillin). See Supplementary data 1 for raw mortality data