Transheterozygous <i>Pten</i>^<i>5</i> mutants exhibit defects in mitochondrial structure in IFM and upregulation of the oxidative stress response gene, <i>GstD1</i>.

<p>(<b>A</b>) qRT-PCR of <i>Pink1</i> mRNA expression levels in third instar larvae carrying different mutations affecting IIS/mTORC1 signalling normalised to wild type <i>w</i>^<i>1118</i> control animals. (<b>B</b>) Levels of the anti-oxidative enzyme-encoding <i>GstD1</i> transcript are elevated significantly in <i>Pten</i> mutant backgrounds compared to <i>w</i>^<i>1118</i> controls. However, there is no significant modulation in the transcript expression levels of <i>GstD1</i> in either <i>foxo</i> or <i>4E-BP</i> mutants or <i>Pten</i> heterozygous animals. Data are presented as mean ± SEM. * <i>P</i> <0.05; ** <i>P</i> < 0.001; ***<i>P</i> < 0.0001, and are from three independent experiments. Significance was determined by one-way ANOVA with Bonferroni post-hoc correction test. (<b>C</b>-<b>H</b>) Longitudinal sections of thoraces of 26-day-old female flies either stained with toluidine blue and visualized by light microscopy (scale bar: 100μm; <b>C</b>,<b>D</b>) or imaged by transmission electron microscopy (TEM; scale bar: 1μm) to visualize ultrastructure of IFMs (<b>E</b>-<b>H</b>). The sarcomeric structure of mutant muscle appears relatively normal (black arrows in <b>F</b>,<b>H</b> compared to controls in <b>E</b>,<b>G</b>), but mitochondrial morphology in the mutant is severely disrupted (white arrows in <b>F</b>,<b>H</b> compared to controls in <b>E</b>,<b>G</b>).</p

Transheterozygous <i>Pten</i>^<i>5</i> mutant flies have a highly penetrant eye phenotype.

<p><b>A-J</b>. Low (<b>A-E</b>) and high (<b>F-J</b>) magnification views of eyes from females of different genotypes. A mild disorganisation of the ommatidia in the posterior region of the eye is observed in <i>Pten</i>^<i>5</i><i>/Pten</i>^<i>1</i> (<b>C</b>,<b>H</b>) and <i>Pten</i>^<i>5</i><i>/Pten</i>^<i>dj189</i> (<b>D</b>, <b>I</b>) flies, as shown by black arrows in <b>H</b> and <b>I</b>, but not in wild type <i>CantonS</i> (<b>A</b>,<b>F</b>) and <i>Pten</i>^<i>5</i> heterozygous control females (<i>Pten</i>^<i>5</i><i>/CyO</i>) (<b>B</b>,<b>G</b>). Almost all female mutant animals carrying a <i>Pten</i> genomic rescue construct (<i>Pten</i>^<i>5</i><i>/Pten</i>^<i>dj189</i><i>;[R]</i>) (<b>E</b>,<b>J</b>) do not display the eye phenotype. (<b>K</b>) Histogram presented as mean percentage of flies exhibiting disorganised eye phenotype. Error bars indicate standard error of mean (SEM). *** <i>P</i> < 0.001, from two separate experiments n ≥ 100. (<b>L</b>) The mean body mass of different <i>Pten</i> mutant females, <i>Pten</i>^<i>5</i><i>/Pten</i>^<i>1</i> and <i>Pten</i>^<i>5</i><i>/Pten</i>^<i>dj189</i>, is not significantly heavier than wild type <i>w</i>^<i>1118</i>. Surprisingly, <i>Pten</i>^<i>5</i><i>/Pten</i>^<i>dj189</i> rescue females have significantly higher body mass than all other genotypes. Data are presented as mean body mass per fly ± SEM. Pooled from two independent experiments, n ≥60. Statistical significance was determined by two-tailed unpaired Student’s <i>t</i>-test. Scale bar: 100μm.</p

<i>Pten</i>^<i>5</i> transheterozygous mutant flies are sensitive to a wide range of stresses.

<p>Survival of <i>Pten</i>^<i>5</i><i>/Pten</i>^<i>dj189</i> transheterozygous mutants (orange), mutants carrying a <i>Pten</i> genomic rescue construct (<i>Pten</i>^<i>5</i><i>/Pten</i>^<i>dj189</i><i>;[R]</i>) (green), and wild type control <i>w</i>^<i>1118</i> (pink) males after they were exposed to (<b>A</b>) 5mM rotenone, (<b>B)</b> 2mM paraquat, (<b>C)</b> water-only diet and <b>(D)</b> 500mM NaCl. The mean survival times (in hours) for <i>Pten</i>^<i>5</i> transheterozygous mutants, rescue flies, and wild type control <i>w</i>^<i>1118</i> flies respectively are: rotenone = 40.4, 89.2 and 107.1; paraquat = 8.6, 24.2, and 50.3; water starvation = 9.2, 19.1 and 24.2; NaCl = 15.4, 19.4 and 30.9. In all four stress assays, <i>Pten</i>^<i>5</i> transheterozygous mutants were short lived compared with wild type <i>w</i>^<i>1118</i> (<i>P</i> < 0.001), and for all but NaCl stress had a significantly shorter mean survival time compared to rescue flies (<i>P</i> < 0.01). For each experiment, flies were grouped into at least 6–8 batches of 20, these experiments were then repeated four times and data pooled together, n ≥ 480). Statistical significance was determined by Mantel-Cox Log rank test and Wilcoxon test using GraphPad5. Graphs presented as pooled data of percentage mean of survival for each genotype. Graphs presented as mean ± SEM.</p

<i>Pten</i>^<i>5</i> transheterozygous mutants exhibit <i>Pten</i>-associated locomotive phenotypes.

<p><b>(A,B)</b> Flight tests of adult females of different genotypes over a 9 day <b>(A)</b> and 25 day <b>(B)</b> period. <b>(A)</b><i>Pten</i>^<i>5</i> transheterozygous mutant female flightlessness rises between 3 and 9 days (***<i>P</i><0.001) and is significantly higher than <i>w</i>^<i>1118</i> and heterozygous <i>Pten</i>^<i>5</i><i>/CyORoi</i> controls over a 9 day period; ***<i>P</i> < 0.001 relative to both controls. There was no statistically significant difference between wild type <i>w</i>^<i>1118</i> and heterozygous control <i>Pten</i>^<i>5</i><i>/CyORoi</i> at any time point; graphs represent pooled data from six experiments, n ≥ 120. <b>(B)</b> Frequency of flightless phenotype for <i>Pten</i>^<i>5</i> transheterozygous mutant female flies continues to increase (<i>P</i> < 0.001 from days 2–9, 9–16 and 16–25 days) and be significantly greater than control <i>w</i>^<i>1118</i> and <i>Pten</i>^<i>5</i><i>/CyORoi</i> females over a 25 day period. Data from at least six independent experiments. **<i>P</i> < 0.01, ***<i>P</i> < 0.001, n ≥ 100, determined by two-way ANOVA with Bonferroni post-hoc correction for <b>A</b> and <b>B</b>. <b>(C)</b> Flightless phenotype in 9-day-old <i>Pten</i>⁵ transheterozygous female flies is strongly rescued by a <i>Pten</i> genomic construct; pooled data from six experiments; *** <i>P</i> < 0.001, n ≥ 100. <b>(D)</b> 9-day-old <i>Pten</i>^<i>5</i> transheterozygous mutant males display a defective geotaxic phenotype compared to <i>w</i>^<i>1118</i> controls, <i>Pten</i>^<i>5</i><i>/CyORoi</i> heterozygotes or genomic rescue flies, assessed by scoring flies that failed to climb 6 cm in 30 sec; n ≥ 50, *** <i>P</i> < 0.001. Significance determined by one-way ANOVA with Bonferroni post-hoc correction for <b>C</b> and <b>D</b>. Graphs present as mean ± SEM.</p

Principal cell ablation increases hemolymph phosphate and decreases life span, rescue by addition of sevelamer to the culture medium.

<p>A, B: Micrographs of 10 days old males with expression of GFP (green) in principal cells (<b>A</b>) or females with expression of GFP along with <i>reaper</i> (<i>rpr</i>) in principal cells after culture for ten days at 29°C (<b>B</b>). Epifluorescent microphotograph (10X) of tubule and gut of the same genotypes shown in A+B. Ablation of GFP-positive cells (green) by <i>rpr</i> is nearly complete with the exception of a few segments as shown schematically in the inset. As a consequence when compared to <i>rpr</i>-negative tubules (<b>C</b>), the majority of the tubule stained with propidium iodide (red) is made up of GFP-negative cells in (<b>D</b>). Median life span (<b>E</b>, n = 60–120 per condition) or hemolymph phosphate concentration (<b>F</b>, n = 3 with collections from 15 flies) of females expressing <i>rpr</i> or <i>GFP</i>-RNAi in principal cells after culture on standard medium containing 30 mM sodium phosphate (P30) normalized when cultured on 1% sevelamer (Sev1%) for 14 days.</p

S2R+ genome-wide RNAi screen. A:

<p>heatmap of z-scores for 146 verified genes, cell count/well based on DAPI signal (first column), dpERK signal after 10 min. phosphate stimulation (middle column), and dpERK signal after 10 min. insulin stimulation (third column), green indicates positive regulators, red indicates negative regulators. <b>B:</b> Functional classification of 146 verified genes based on GO term categories using the DAVID tool (<a href="http://david.abcc.ncifcrf.gov/" target="_blank">http://david.abcc.ncifcrf.gov/</a>) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0056753#pone.0056753-Huangda1" target="_blank">[27]</a>, and FlyMine (<a href="http://www.flymine.org/" target="_blank">www.flymine.org/</a>) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0056753#pone.0056753-Lyne1" target="_blank">[28]</a>. <b>C:</b> Number of genes identified in prior DRSC screens.</p

RNAi-mediated inhibition of MAPK-signaling <i>in vivo</i> decreases hemolymph phosphate. A:

<p>The most advanced developmental stage with induced knockdown was scored on standard medium (1 = embryonic, 2 = first and second instar larva, 3 = third instar larva, 4 = pupal lethal, 4.5 = developmental delay, adult, 5 = adult). <b>B:</b> hemolymph phosphate concentration of young adult females cultured at 29°C for five days, and <b>C:</b> median life span of adult males cultured at 29°C on standard medium (see also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0056753#pone.0056753.s010" target="_blank">Table S1</a>). <b>a:</b> p<0.05, <b>b:</b> p<0.007 vs. Luc/GFP/white controls. P<0.017 was used to test for multiple comparisons between three treatments.</p

Phosphate supplementation or inhibitors of phosphate uptake influences adult life span.

<p>Median life span of adult <i>y w</i> males cultured on standard medium (SM): Control (C, n = 550), 30 mM sodium phosphate (P30, n = 465), 30 mM sodium sulfate (S30, n = 245), 1% sevelamer (Sev1, n = 207), 1 mM phosphonoformic acid (PFA1, n = 130), and the combinations: Sev1+P30 (n = 115), PFA1+P30 (n = 137), or defined medium (DM) supplemented with 1.5, 27, or 53 mM sodium phosphate (P1.5, n = 202; P27, n = 200; P53, n = 194). P<0.005 was used to test for multiple comparisons between eleven treatments.</p

Significant outliers in development, life span, and hemolymph phosphate.

<p><i>Drosophila</i> gene names for developmental lethal mutants and outliers in life span and hemolymph phosphate assays shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0056753#pone-0056753-g006" target="_blank">Figures 6</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0056753#pone-0056753-g007" target="_blank">7</a>. Significance for longevity and hemolymph phosphate is based on Student’s t-test (p<0.05). Bold script indicates genes that remain significant after Bonferroni’s correction for multiple comparisons when we used p<0.003 for the life span assay based on 17 outliers and when we used p<0.007 and p<0.0125 for the hemolymph assay based on seven and four outliers, respectively. Underline script is used for longer/higher outliers, while regular script is used for shorter/lower outliers. (See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0056753#pone.0056753.s010" target="_blank">Table S1</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0056753#pone-0056753-g007" target="_blank">Figs. 7</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0056753#pone-0056753-g008" target="_blank">8</a> for detailed results).</p

Adult hemolymph phosphate, phosphate excretion and whole fly Pi. A:

<p>Excretion of phosphate after culture of <i>y w</i> females for five days on standard medium (C) alone or supplemented with 1% sevelamer (Sev1%) and 30 mM sodium phosphate (P30) (n = 3 pooled collections of 15–20 flies). <b>B:</b> hemolymph phosphate concentration (n = 3 pooled collections of 15 flies) and <b>C:</b> whole fly phosphate (n = 10) of flies cultured as described for A. Note that <i>y w</i> and CS flies have lower hemolymph phosphate concentrations than OR and <i>w¹¹¹⁸</i> flies and the F1 generation females that express control RNAis (see Methods and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0056753#pone.0056753.s005" target="_blank">Fig. S5</a>), which is likely due to differences in genetic background.</p