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Science 23 December 2005:
Vol. 310. no. 5756, pp. 1954 - 1957
DOI: 10.1126/science.1115596
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Reports

A Developmental Timing MicroRNA and Its Target Regulate Life Span in C. elegans

Michelle Boehm and Frank Slack*

Department of Molecular, Cellular, and Developmental Biology, Yale University, New Haven, CT 06511, USA.


Fig. 1. lin-4 and lin-14 mutants have opposite life-span phenotypes. (A) Red, survival of wild-type (N2) animals on control bacteria containing empty vector (mock RNAi); blue, lin-4(e912)lf; mock RNAi; pink, lin-14(RNAi); light blue, lin-4(e912)lf; lin-14(RNAi) at 20°C. N2: n = 69, m = 14.6. lin-4(e912)lf: n = 56, m = 6.9, P < 0.0001*. N2;lin-14(RNAi): n = 68, m = 18.7, P < 0.0001*. lin-4(e912)lf; lin-14(RNAi): n = 72, m = 16.6, P < 0.0001#. (B) A lin-14(lf) mutation extends life span when grown and assayed at the restrictive temperature of 25°C. N2: n = 57, m = 9.5. lin-14(n179)lf: n = 58, m = 12.5, P < 0.0001*. lin-4(e912)lf; lin-14(n179)lf: n = 51, m = 9.0, P = 0.0906*. (C) lin-4 overexpression extends life span. Three lines overexpressing (o/e) lin-4 are shown in purple, blue, and green; wild-type animals are in red. N2: n = 64, m = 15.8. lin-4 o/e line 3.3: n = 54, m = 18.3, P < 0.0001*. lin-4 o/e line 3.14: n = 62, m = 17.7, P = 0.0023*. lin-4 o/e line 4.9: n = 37, m = 17.8, P = 0.0113*. (D) A lin-14 gain-of-function mutant, n355, has a short-lived phenotype similar to that of the lin-4(e912)lf mutant. Red, wild-type animals; green, lin-4(e912)lf; blue, lin-14(n355 gf). N2: n = 59, m = 15.9. lin-4(e912)lf: n = 85, m = 7.7, P < 0.0001*. lin-14(n355 gf): n = 94, m = 5.9, P < 0.0001*. All experiments were repeated at least once with similar effects. n, number of animals observed in each experiment. m, mean adult life span (days). P* values refer to experimental strain and N2 control animals in a single experiment, and P# values refer to a strain on control and experimental RNAi treatment in a single experiment. [View Larger Version of this Image (26K GIF file)]
 

Fig. 2. lin-4 and lin-14 mutants display accelerated and delayed rates, respectively, of lipofuscin accumulation. (A) lin-4(e912)lf mutants display an increase of lipofuscin as compared with similarly aged wild-type animals at 20°C. This effect is suppressed by the lin-14(n179)lf mutation. (B) Quantification of the N2, lin-4(e912)lf, lin-4(e912)lf;lin-14(n179)lf populations' gut autofluorescence at days 0, 4, 8, and 12 after the larval-to-adult transition at 20°C. (C) lin-14(n179)lf mutants display a decrease in lipofuscin accumulation compared with wild-type animals at 25°C. (D) Quantification of the N2 and lin-14(n179)lf populations' gut autofluorescence at 25°C as for (B). For (A) and (C), photographs shown are representative examples (n = 10 for each time point per strain). Photographs were taken at 100x magnification. All animals were photographed on the same day under identical conditions, and photographs were treated identically. For (B) and (D), autofluorescence was quantified using Axiovision 4.4 software (n = 10 for each time point per strain). P values were calculated using the Mann-Whitney nonparametric t test comparing mutant to wild-type results at day 12 in (B) and day 8 in (D). Error bars represent the standard error of the mean. [View Larger Version of this Image (61K GIF file)]
 

Fig. 3. Loss of lin-14 function during adulthood is sufficient to extend life span. (A) Wild-type animals treated with lin-14(RNAi) (pink) only in the adult stage have extended life spans compared with mock RNAi animals (red). lin-4(e912)lf mutants treated with lin-14(RNAi) (light blue) only in the adult stage display an extended life span compared with lin-4(e912)lf mutants on mock RNAi (blue) at 20°C. N2: n = 55, m = 14.8. lin-14(RNAi): n = 55, m = 17.6, P < 0.0001*. lin-4(e912)lf: n = 43, m = 9.0. lin-4(e912)lf; lin-14(RNAi): n = 40, m = 12.1, P < 0.0001#. (B) lin-14(n179)lf animals (blue) display extended life spans compared with wild-type animals (red) when grown at the permissive temperature of 15°C until the larval-to-adult transition and then moved to the restrictive temperature of 25°C. N2: n = 48, m = 10.7. lin-14(n179)lf: n = 53, m = 12.4, P < 0.0001*. All experiments were repeated at least once with similar effects. P* and P# are defined in the legend to Fig. 1. [View Larger Version of this Image (24K GIF file)]
 

Fig. 4. The life-span extension of a lin-14(lf) mutant is daf-16 dependent. (A) The life-span extension conferred by the lin-14(n179)lf mutation is abolished with daf-16(RNAi) (light green); daf-16(RNAi) animals (green—obscured by light green). lin-14(lf); daf-2(RNAi) (light blue) displays a further lengthening of the life-span extension conferred by daf-2(RNAi) (blue) at 25°C. Wild-type (red) and lin-14(n179)lf (pink) animals on mock RNAi are shown for comparison. N2: n = 60, m = 8.9. lin-14(n179)lf: n = 56, m = 11.9, P < 0.0001*. daf-2(RNAi): n = 58, m = 17.1. lin-14(n179)lf; daf-2(RNAi): n = 56, m = 23.0, P < 0.0001*. daf-16(RNAi): n = 55, m = 8.3. lin-14(n179)lf; daf-16(RNAi): n = 54, m = 8.4, P = 0.9330*. (B) lin-14(RNAi) is unable to extend the life span of daf-16(mu86) (light blue) or daf-2(e1370)lf (light green) mutants as compared with these strains grown on mock RNAi (blue and green, respectively) at 15°C. Wild-type animals on mock RNAi (red) and on lin-14(RNAi) (pink) are shown for comparison. N2: n = 45, m = 21.6. lin-14(RNAi): n = 51, m = 25.1, P = 0.0003*. daf-2(e1370)lf: n = 49, m = 32.3. daf-2(e1370)lf; lin-14(RNAi): n = 53, m = 31.1, P = 0.0100#. daf-16(mu86): n = 28, m = 16.4. daf-16(mu86); lin-14(RNAi): n = 42, m = 16.7, P = 0.2300#. (C) A wild-type copy of lin-4 is required for full life-span extension by daf-2(RNAi) (blue versus light blue), and is also required for the life-span phenotype conferred by daf-16(RNAi) (purple versus light purple). Wild-type (red) and lin-4(e912)lf (pink) animals grown on mock RNAi are shown for comparison. N2: n = 70, m = 16.0. lin-4(e912)lf: n = 52, m = 6.8, P < 0.0001*. daf-2(e1370): n = 67, m = 25.5. lin-4(e912)lf; daf-2(RNAi): n = 55, m = 13.8, P < 0.0001*. daf-16(RNAi): n = 64, m = 10.7. lin-4(e912)lf; daf-16(RNAi): n = 55, m = 6.1, P < 0.0001*. All experiments were repeated at least once with similar results. P* and P# are defined in the legend to Fig. 1. [View Larger Version of this Image (27K GIF file)]
 

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