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. 2013 Aug 29;9(8):e1003737. doi: 10.1371/journal.pgen.1003737

Figure 2. mir-80(Δ) exhibits multiple characteristics typical of DR animals.

Figure 2

Fig. 2A. The mir-80(Δ) mutant exhibits the DR Exmax shift. We assayed age-synchronized WT (black), mir-80(Δ) (red), and mir-80(Δ); Ex[Pmir-80(+)] (grey) 4 day old animals grown under standard conditions (20°C, OP50-1). We used a spectrofluorimeter to scan transparent animals (n = 100 per strain/trial) to generate excitation/emission profiles as in [19]; wavelength of excitation at maximal fluorescence is indicated. Graphs represent mean data from at least 3 independent trials. Data were compared One-way ANOVA followed by Newman-Keuls multiple comparison test, *** - p<0.0005. mir-80(Δ) exhibits a significantly down-shifted Exmax (p<0.0005) as compared to wild type under conditions of abundant food, a feature unique to DR [19]. Fig. 2B. The mir-80(Δ) mutant exhibits low age pigment levels early in life, as occurs in C. elegans DR. We assayed age-synchronized WT (black), mir-80(Δ) (red), and mir-80(Δ); Ex[Pmir-80(+)] (grey) 4 day old animals grown under standard conditions (20°C, OP50-1). We scanned animals (n = 100 per strain) for fluorescence over a range of wavelengths, and normalized age pigment fluorescence (AGE) to tryptophan (TRP) fluorescence as in [19] for comparison. Graphs represent mean data from at least 3 independent trials. Data were compared using One-way ANOVA followed by Newman-Keuls multiple comparison test, *** - p<0.0005, ** - p<0.005. mir-80(Δ) exhibits low age pigment levels as compared to wild type (p<0.0005) early in life, which is true of all DR conditions previously tested (although not unique to DR). In these assays, levels were on average 66% lower in mir-80(Δ). Fig. 2C. mir-80(Δ) mutants are physically smaller than WT, typical of animals in DR. We measured age-synchronized WT (black) and mir-80(Δ) (red) 4 day old animals (examples at the right) grown under standard conditions (20°C, OP50-1) by imaging animals (WT n = 77, mir-80(Δ) n = 88) under DIC under low magnification. We measured using the segmented line tool in the ImageJ software by drawing a line across the length of the animal, and converted length in pixels to uM using a stage micrometer to assess image scale. We compared data using 2-tailed Student's T-test, *** - p<0.0005. mir-80(Δ) mutants are ∼10% shorter and look thinner than WT reared under the same conditions, typical of the scrawny appearance of animals in DR, example comparison on the right. Although size varies somewhat and is not as quantitative a measure as age pigment scores, we have used the scrawny appearance to identify likely mir-80(Δ) homozygotes in crosses. Fig. 2D. mir-80(Δ) mutants exhibit reduced fertility and an extended reproductive lifespan. We assayed egg production in age-synchronized WT (black), mir-80(Δ) (red), and DR mutant eat-2(ad1116) (blue) grown under standard conditions (20°C, OP50-1; parent n = 10, 3 independent trials). eat-2 is one trial so bars are not provided. Data were compared using 2-tailed Student's T-test. Early in adult life, mir-80(Δ) produce a reduced number of live births per day (33% decrease, p<0.05 for Day 3; 180% increase, p<0.001 Day 4–6) and exhibit a prolonged reproductive lifespan (through Day 8 for mir-80(Δ) as compared to WT Day 6, p<0.001). The constitutive DR mutant, eat-2 experiences a shift in reproductive lifespan (compared to WT, p<0.001) that is similar to mir-80(Δ). Fig. 2E. SKN-1-GFP, a molecular reporter of DR, is upregulated in mir-80(Δ) in the presence of food. SKN-1::GFP expression in the two ASI neurons is a molecular signal of some DR [7]. We constructed strains that included an integrated rescuing skn-1-gfp fusion gene expressed from the native skn-1 promoter, Is007[skn-1-gfp] [7], and measured at Day 7, 20°C, growth in OP50-1 (white arrows). WT animals show low levels of ASI expression (36% with very weak expression in only one ASI), while DR-constitutive eat-2(ad1116) animals display constitutive expression of SKN-1-GFP in the ASI neurons (92% in 1 or 2 neurons, strong expression). 95% of mir-80(Δ) have 1–2 ASIs expressing at this timepoint. These data support that mir-80(Δ) mutants are in DR even when reared in the presence of ample food.