NHR-49 Helps Germline-Less Worms Chew the Fat

In C. elegans, removal of the germline extends lifespan significantly. We demonstrate that the nuclear hormone receptor, NHR-49, enables the response to this physiological change by increasing the expression of genes involved in mitochondrial β-oxidation and fatty-acid desaturation. The coordinated augmentation of these processes is critical for germline-less animals to maintain their lipid stores and to sustain de novo fat synthesis during adulthood. Following germline ablation, NHR-49 is up-regulated in somatic cells by the conserved longevity determinants DAF-16/FOXO and TCER-1/TCERG1. Accordingly, NHR-49 overexpression in fertile animals extends their lifespan modestly. In fertile adults, nhr-49 expression is DAF-16/FOXO and TCER-1/TCERG1 independent although its depletion causes age-related lipid abnormalities. Our data provide molecular insights into how reproductive stimuli are integrated into global metabolic changes to alter the lifespan of the animal. They suggest that NHR-49 may facilitate the adaptation to loss of reproductive potential through synchronized enhancement of fatty-acid oxidation and desaturation, thus breaking down some fats ordained for reproduction and orchestrating a lipid profile conducive for somatic maintenance and longevity

Germline signals deploy NHR-49 to modulate fatty-acid β-oxidation and desaturation in somatic tissues of C. elegans.

In C. elegans, removal of the germline extends lifespan significantly. We demonstrate that the nuclear hormone receptor, NHR-49, enables the response to this physiological change by increasing the expression of genes involved in mitochondrial β-oxidation and fatty-acid desaturation. The coordinated augmentation of these processes is critical for germline-less animals to maintain their lipid stores and to sustain de novo fat synthesis during adulthood. Following germline ablation, NHR-49 is up-regulated in somatic cells by the conserved longevity determinants DAF-16/FOXO and TCER-1/TCERG1. Accordingly, NHR-49 overexpression in fertile animals extends their lifespan modestly. In fertile adults, nhr-49 expression is DAF-16/FOXO and TCER-1/TCERG1 independent although its depletion causes age-related lipid abnormalities. Our data provide molecular insights into how reproductive stimuli are integrated into global metabolic changes to alter the lifespan of the animal. They suggest that NHR-49 may facilitate the adaptation to loss of reproductive potential through synchronized enhancement of fatty-acid oxidation and desaturation, thus breaking down some fats ordained for reproduction and orchestrating a lipid profile conducive for somatic maintenance and longevity

NHR-49 overexpression increases the lifespan of fertile worms.

<p><b>A</b>: The levels of <i>nhr-49</i> mRNA compared using Q-PCRs between wild-type (N2, gray), <i>nhr-49</i> mutants (blue) and worms overexpressing NHR-49 through the NHR-49::GFP transgene (<i>NHR-49 OE</i>) in these two genetic backgrounds (maroon and red, respectively). The X-axis represents the strains being compared and the Y-axis the fold change in expression. The data is combined from four independent biological replicates, each with three technical replicates. Error bars display standard error of the mean, and asterisks depict the statistical significance of the differences observed in an unpaired, two-tailed t-test. N2 <i>vs. NHR-49 OE</i> P = 0.0006 (maroon asterisks); <i>nhr-49 vs. nhr-49</i>;<i>NHR-49 OE</i> P = 0.002 (red asterisks); N2 <i>vs. nhr-49</i> P = 0.002 (blue asterisks). <b>B–C: Effects of NHR-49 overexpression on lifespan of fertile worms.</b><b>B:</b> N2 (black; m = 21.6±0.1, n = 75/98), <i>nhr-49</i> (purple; m = 14.4±0.2, n = 89/100, P <i>vs.</i> N2<0.0001), <i>NHR-49 OE</i> non-transgenic siblings (gray; m = 21.3±0.4, n = 83/102, P <i>vs.</i> N2 0.82), <i>NHR-49 OE</i> (red; m = 26.0±0.4, n = 47/84, P <i>vs.</i> N2<0.0001, P <i>vs. nhr-49</i><0.0001, P <i>vs.</i> non-transgenic siblings <0.0001). <b>C:</b> N2 (black; m = 22.8±0.2, n = 81/92), <i>nhr-49</i> (purple; m = 13.7±0.1, n = 83/88, P <i>vs.</i> N2<0.0001), <i>nhr-49;NHR-49 OE</i> non-transgenic siblings (gray; m = 12.4±0.1, n = 93/98, P <i>vs. nhr-49</i> 0.02, P <i>vs.</i> N2<0.0001), <i>nhr-49;NHR-49 OE</i> (red; m = 25.1±0.3, n = 79/101, P <i>vs.</i> N2<0.0001, P <i>vs. nhr-49</i><0.0001, P <i>vs.</i> non-transgenic siblings <0.0001). <b>D: Effect of NHR-49 overexpression on lifespan of </b><b><i>glp-1</i></b><b> mutants.</b> N2 (black; m = 21.6±0.1, n = 75/98), <i>glp- 1</i> (green; m = 31.0±0.5, n = 94/101, P <i>vs.</i> N2<0.0001), <i>nhr-49;glp-1</i> (purple; m = 14.1±0.1, n = 95/97, P <i>vs. glp-1</i><0.0001), <i>glp-1;NHR-49 OE</i> (red; m = 35.3±0.5, n = 92/97, P <i>vs. glp-1</i> 0.001). <b>E, F:</b><b>Effect of </b><b><i>daf-16</i></b><b> and </b><b><i>tcer-1</i></b><b> reduction of function on lifespan extended by NHR-49 overexpression. E:</b> N2 (black; m = 18.5±0.3, n = 67/109), <i>nhr-49</i> (purple; m = 12.7±0.1, n = 87/96, P <i>vs.</i> N2<0.0001), <i>daf-16 (</i>blue; 15.6±0.2, n = 56/120, P <i>vs.</i> N2 0.001), <i>NHR-49 OE</i> (green; m = 24.9±0.2, n = 40/98, P <i>vs.</i> N2<0.0001), <i>daf-16;NHR-49 OE</i> (red; m = 17.4±0.3, n = 56/96, P <i>vs.</i> N2 0.26, P <i>vs. daf-16</i> 0.26, P <i>vs. nhr-49</i><0.0001). <b>F:</b> N2 worms grown on control vector bacteria (black; m = 20.3±0.7, n = 59/63). <i>NHR-49 OE</i> worms grown on control vector bacteria (green; m = 21.6±1.4; n = 21/71; P vs N2 on control vector 0.05), <i>daf-16</i> RNAi bacteria (red; m = 11.8±0.3; n = 39/62; P <i>vs.</i> control <0.0001) and on <i>tcer-1</i> RNAi bacteria (blue; m = 14.1±0.7; n = 40/64; P <i>vs.</i> control <0.0001). Additional control lifespans not shown in the graph: N2 worms grown on <i>daf-16</i> RNAi bacteria (m = 15.1±0.4; n = 59/63; P <i>vs.</i> N2 control <0.0001) and on <i>tcer-1</i> RNAi bacteria (m = 20.2±0.2; n = 53/65; P <i>vs.</i> N2 control 0.4). Data from additional trials is presented in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004829#pgen.1004829.s015" target="_blank">S3 (B–D)</a> and <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004829#pgen.1004829.s017" target="_blank">S5A (E) Tables</a>.</p

NHR-49 is required for maintenance of fat stores and <i>de novo</i> fat synthesis in germline-less adults.

<p><b>A</b>–<b>I: </b><b><i>nhr-49</i></b><b>;</b><b><i>glp-1</i></b><b> mutants undergo a dramatic depletion of lipid stores during young adulthood.</b> Lipid levels compared between <i>glp-1</i> (A, C, E and G) and <i>nhr-49</i>; <i>glp-1</i> (B, D, F and H) through ORO staining of L4 larvae (day 0) and adults on days 1 (A, B), 2 (C, D), 4 (E, F), 6 (G, H) and 8 (I). Representative images are shown in A-H and the quantification of the data is in I. The two strains show similar fat levels on Day 1, but by day 8 the <i>nhr-49</i>;<i>glp-1</i> mutants exhibit a significant reduction in ORO staining. <b>J: </b><b><i>nhr-49</i></b><b>;</b><b><i>glp-1</i></b><b> mutants show decreased TAG levels.</b> Using GC/MS, the triglyceride: phospholipid (TAG/PL) ratio of day 2 <i>nhr-49</i>;<i>glp-1</i> adults was found to be significantly lesser than that of age-matched <i>glp-1</i> animals. <b>K: </b><b><i>de novo</i></b><b> fatty-acid synthesis is impaired in </b><b><i>nhr-49</i></b><b>;</b><b><i>glp-1</i></b><b> mutants.</b> Using a 13C isotope fatty-acid labeling assay, <i>de novo</i> fat synthesis and dietary fat absorption were compared between day 2 <i>glp-1</i> and <i>nhr-49</i>;<i>glp-1</i> adults. Individual fatty-acid species are represented on the X-axis and relative synthesis levels are on the Y-axis. Synthesis of six out of seven species was significantly reduced upon <i>nhr-49</i> reduction of function. <b>L: </b><b><i>nhr-49</i></b><b> mutants undergo lipid depletion with age.</b> Lipid levels compared between wild-type (N2, gray), <i>nhr-49</i> (blue), <i>NHR-49::GFP</i> (<i>NHR-49 OE,</i> purple) and <i>nhr-49;NHR-49::GFP</i> (<i>nhr-49;NHR-49 OE,</i> brown) strains through ORO staining of adults on days 2, 4 and 6 of adulthood. The strains show similar fat levels on day 2, but by day 6 <i>nhr-49</i> mutants as well as worms overexpressing NHR-49 display a significant reduction in ORO staining. <b>M: </b><b><i>nhr-49</i></b><b> mutants do not have increased TAGs.</b> Using GC/MS, the triglyceride: phospholipid (TAG/PL) ratio of late L4/early day 1 <i>nhr-49</i> mutants was found to be similar to that of age-matched wild-type animals. <b>N: </b><b><i>de novo</i></b><b> fatty-acid synthesis is disrupted in </b><b><i>nhr-49</i></b><b> mutants.</b><i>de novo</i> fat synthesis and dietary fat absorption were compared between late L4/early day 1 <i>nhr-49</i> mutants and wild-type (N2) adults using the 13C isotope fatty-acid labeling assay. Synthesis of some fatty acids was reduced and that of others was increased in <i>nhr-49</i> mutants in the neutral lipid fraction (see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004829#pgen.1004829.s008" target="_blank">S8 Figure</a> for phospholipid data). A similar comparison of age-matched <i>glp-1</i> and <i>nhr-49</i>;<i>glp-1</i> adults presented a similar mixed profile with the notable exception of OA whose synthesis was reduced in <i>nhr-49;glp-1</i> mutants at both stages while in <i>nhr-49</i> mutants it was synthesized at a higher level (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004829#pgen.1004829.s008" target="_blank">S8B, C Figure</a>). By day 2, the synthesis of all fatty acids tested was uniformly reduced in <i>nhr-49</i>; <i>glp-1</i> mutants (K). All graphs were obtained by combining data from at least three independent biological replicates. Error bars indicate the standard error of the mean. Asterisks depict the statistical significance of the observed differences in an unpaired, two-tailed t-test with P values 0.05 (*), 0.005 (**) and <0.0001 (***). The color of the asterisk denotes the strain showing the observed reduction.</p

Multiple mitochondrial fatty-acid β-oxidation genes regulated by NHR-49 are essential for the increased longevity of germline-less animals but not wild-type worms.

<p><i>glp-1</i> mutants (1A) and the sterile strain, <i>fer-15(b26);fem-1(hc17),</i> (1B) were subjected to ‘adult-only’ RNAi inactivation of mitochondrial <b>β-</b>oxidation genes whose expressions were elevated upon germline loss, many in an NHR-49-dependent manner. See <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004829#s4" target="_blank">methods</a> section for experimental details and <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004829#pgen.1004829.s018" target="_blank">S6 Table</a> for additional trials.</p><p>*<i>fer-15(b26);fem-1(hc17)</i> is a temperature sensitive strain that when grown at 25°C is sterile and used as a surrogate for wild-type, N2 in lifespan assays <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004829#pgen.1004829-Amrit2" target="_blank">[69]</a>.</p><p>**<i>ech-1.2</i> (T08B2.7) is orthologous to the human gene that encodes the tri-functional protein, hydroxyl acyl CoA dehydrogenase/3-ketoacyl CoA thiolase/enoyl CoA hydratase (HADHA) alpha subunit (<a href="http://www.wormbase.org" target="_blank">www.wormbase.org</a>) and a close paralog of <i>ech-1.1</i>. <i>ech-1.1</i> and <i>cpt-5</i> could not be tested due to contamination of RNAi clones. Data is shown as mean lifespan in days (Mean) ± standard error of the mean (SEM). ‘n’ refers to the number of worms observed (obs) divided by total number of worms tested in the experiment.</p>a<p>some worms were censored from the analysis as described in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004829#s4" target="_blank">methods</a>.</p>b<p>Empty vector control refers to worms exposed to empty vector plasmid without an RNAi insert. P values were calculated using the log rank (Mantel Cox) method.</p><p>Multiple mitochondrial fatty-acid β-oxidation genes regulated by NHR-49 are essential for the increased longevity of germline-less animals but not wild-type worms.</p

Germline removal causes increased expression of NHR-49 under regulation of DAF-16 and TCER-1.

<p><b>A</b>–<b>E: Elevation of NHR-49::GFP in germline-less animals by DAF-16 and TCER-1.</b> NHR-49::GFP fluorescence observed in wild type (N) worms (A) (n = 219) and <i>glp-1</i> (B) (n = 383), <i>daf-16;glp-1</i> (C) (n = 175) and <i>tcer-1;glp-1</i> (D) (n = 267) mutants. The increased GFP in <i>glp-1</i> is visible in intestinal nuclei (compare A and B) and is abolished in <i>daf-16</i>;<i>glp-1</i> animals (C). <i>tcer-1</i>;<i>glp-1</i> mutants exhibit high expression in some gut cells but no GFP in others (D). The bar graph in E shows the quantification of these data obtained from day 2 young adults of each strain classified into those with high (green), medium (peach), and low (gray) GFP. In the <i>tcer-1</i>;<i>glp-1</i> bar, the high GFP class is shown as spotted green to indicate that these worms showed high but mosaic intestinal expression. <b>F: Selective effect of </b><b><i>daf-16</i></b><b> and </b><b><i>tcer-1</i></b><b> RNAi on NHR-49::GFP in </b><b><i>glp-1</i></b><b> mutants.</b> NHR-49::GFP fluorescence in wild-type animals (N2, solid bars) and in <i>glp-1</i> background (striped bars) observed in day 2 adults. Worms were grown on bacteria containing empty control vector (EV) or those expressing dsRNA targeting <i>daf-16, tcer-1</i> or <i>nhr-49</i>. GFP classification is the same as in E. <i>daf-16</i> or <i>tcer-1</i> RNAi treatments suppress the increased GFP seen in <i>glp-1</i> mutants, but not in wild-type worms (both strains were tested simultaneously). In the N2 background, n = 175, 113, 136 and 64, respectively for EV, <i>daf-16, tcer-1</i> or <i>nhr-49</i> RNAi, respectively. In the glp-1 background, n = 206, 146, 202 and 81, respectively for EV, <i>daf-16, tcer-1</i> or <i>nhr-49</i> RNAi, respectively. In E and F, ‘n’ signifies the total number of worms examined in three-to-five independent trials. <b>G, H: The control of </b><b><i>nhr-49</i></b><b> mRNA levels by DAF-16 and TCER-1 in fertile </b><b><i>vs.</i></b><b> germline-less adults.</b> Q-PCR analysis used to compare the mRNA levels of <i>nhr-49</i> between wild type (N2), <i>glp-1, daf-16;glp-1</i> and <i>tcer-1;glp-1</i> day 2 adults grown under similar conditions (G) as well as day 2 adults of N2, <i>nhr-49, daf-16</i> and <i>tcer-1</i> single mutants (H). Strains are represented on the X-axis and relative expression levels are on the Y-axis. The asterisks represent the statistical significance of the differences in expression in an unpaired, two-tailed t-test with P values 0.05 (*) and 0.005 (**). Error bars in E–H represent the standard error of the mean. In G, the difference between <i>glp-1</i> and <i>tcer-1;glp-1</i> was statistically significant in four of seven biological replicates (each with three technical replicates), but did not achieve significance when data from all the trials were combined.</p

NHR-49 up-regulates the expression of genes involved in different steps of mitochondrial β-oxidation upon germline loss.

<p><b>A, B: Schematic representation of the mitochondrial β-oxidation, fatty-acid desaturation and elongation pathways.</b> Free fatty acids can be directed for breakdown through β-oxidation in peroxisomes and mitochondria (A) or take an anabolic path through desaturation and elongation (B). <b>A</b>: Mitochondrial β-oxidation involves the repetitive action of a series of enzymes that ultimately results in the breakdown of fatty acids into acetyl CoA <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004829#pgen.1004829-Houten1" target="_blank">[43]</a>. Enzymes that function at each of these stages are indicated in purple boxes; ACS (acyl CoA synthetase), CPT (carnitine palmitoyl transferase), ACDH (acyl CoA dehydrogenase), ECH (enoyl CoA hydratase), HACD (hydroxyl acyl CoA dehydrogenase) and thiolase. The genes encoding these enzymes that were up-regulated in <i>glp-1</i> mutants are represented in green. Of these, those dependent on <i>nhr-49</i> for their up-regulation are in bold. Genes repressed by <i>nhr-49</i> are in red. The dashed lines mark the mitochondrial membranes. <b>B</b>: The steps involved in fatty-acid desaturation and elongation that results in conversion of small SFAs to longer MUFA and PUFA species are depicted through the example of palmitic acid (C16:0) <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004829#pgen.1004829-VanGilst1" target="_blank">[36]</a>. Genes up-regulated in <i>glp-1</i> mutants by <i>nhr-49</i> are highlighted in green and those repressed are in red. Fatty-acid species that showed significantly different levels between day 2 <i>glp-1</i> and <i>nhr-49</i>;<i>glp-1</i> mutants in GC/MS are represented in color: fatty acids reduced in <i>nhr-49</i>; <i>glp-1</i> mutants are in blue and those elevated are in pink. Some PUFAs were elevated only in the phospholipid fraction of <i>nhr-49</i>;<i>glp-1</i> mutants and these are highlighted in purple. For a complete list of genes involved in both these pathways see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004829#pgen.1004829.s004" target="_blank">S4 Figure</a>. <b>C</b>–<b>N: The expression of multiple mitochondrial β-oxidation genes is elevated in germline-depleted animals under control of </b><b><i>nhr-49</i></b><b>.</b> mRNA levels of mitochondrial β-oxidation genes examined by Q-PCRs performed on at least three biological replicates isolated from day 2 adults of the following strains: wild-type (N2, gray), <i>nhr-49</i> (blue), <i>glp-1</i> (green) and <i>nhr-49</i>;<i>glp-1</i> (red). All 12 genes tested showed increased expression in <i>glp-1</i> mutants. Of these, the up-regulation of 7 genes was significantly reduced or abolished in <i>nhr-49</i>;<i>glp-1</i> mutants (effect on <i>ech-7</i> did not achieve statistical significance). These included previously identified NHR-49 targets {<i>acs-2</i> (B), <i>cpt-5</i> (J), <i>ech-1.1</i> (D) and <i>hacd-1</i> (E)} as well as new ones {<i>acs-22</i> (D), <i>acdh-11</i> (N)}. <i>acdh-9</i> mRNA was elevated in <i>glp-1</i> mutants compared to N2 but further elevated in <i>nhr-49</i>;<i>glp-1</i> mutants (F). The statistical significance of the N2 <i>vs. glp-1</i>, <i>glp-1 vs. nhr-49</i>;<i>glp-1</i> and N2 <i>vs. nhr-49</i> comparisons in an unpaired, two-tailed t-test are represented by green, red and blue asterisks, respectively. Number of asterisks correspond to P values 0.05 (*), 0.005 (**) and <0.0001 (***).</p

NHR-49 is essential for the longevity of germline-depleted animals and is widely expressed in somatic cells.

<p><b>A: Effect of <i>nhr-49</i> RNAi on the lifespan of germline defective <i>glp-1</i> adults.</b><i>glp-1</i> mutants were subjected to RNAi during adulthood by feeding bacteria containing control (empty) vector (green; m = 26.3±0.6, n = 92/96) as well as bacteria expressing dsRNA targeting <i>daf-16</i> (blue; m = 16.4±0.2, n = 92/97; P <i>vs.</i> control <0.0001), <i>nhr-49</i> RNAi clone #1 (red; m = 17.3±0.3, n = 99/101; P <i>vs.</i> control <0.0001, P <i>vs. daf-16</i> RNAi 0.01) and <i>nhr-49</i> RNAi clone #2 (maroon; m = 15.5±0.1, n = 79/92, P <i>vs.</i> control, <0.0001, P <i>vs. daf-16</i> RNAi 0.005). Clones #1 and #2 were obtained from the Ahringer and Vidal feeding RNAi libraries <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004829#pgen.1004829-Rual1" target="_blank">[39]</a>, <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004829#pgen.1004829-Kamath1" target="_blank">[40]</a>, respectively. <b>B: Effect of </b><b><i>nhr-49</i></b><b> mutation on the lifespan of </b><b><i>glp-1</i></b><b> mutants and wild-type (N2) worms.</b><i>glp-1</i> (green; m = 31.0±0.5, n = 94/101), <i>nhr-49;glp-1</i> (red; m = 14.1±0.1, n = 95/97; P <i>vs. glp-1</i><0.0001), N2 (black; m = 21.6±0.1, n = 75/98), <i>nhr-49</i> (brown; m = 14.4±0.2, n = 89/100; P <i>vs.</i> N2<0.0001). <b>C: Effect of </b><b><i>nhr-49</i></b><b> mutation on the lifespan of </b><b><i>daf-2</i></b><b> mutants.</b> N2 (black; m = 16.9±0.1, n = 48/80), <i>nhr-49</i> (brown; m = 10.7±0.1, n = 80/91; P <i>vs.</i> N2<0.0001). <i>daf-2(e1368)</i> (blue; m = 30.6±0.4, n = 31/75; P <i>vs.</i> N2<0.0001), <i>nhr-49(nr2041);daf-2(e1368)</i> (pink; m = 29.5±0.7, n = 45/86; P <i>vs.</i> N2<0.0001; P <i>vs. daf-2(e1368)</i> 0.73). <i>daf-2(e1370)</i> (green; m = 43.0±0.6, n = 42/67; P <i>vs.</i> N2<0.0001), <i>nhr-49(nr2041);daf-2(e1370)</i> (red; m = 42.4±0.6, n = 46/74; P <i>vs.</i> N2<0.0001; P <i>vs. daf-2(e1370)</i> 0.004). <b>D: Effect of </b><b><i>cyc-1</i></b><b> RNAi on lifespan of N2 and </b><b><i>nhr-49</i></b><b>.</b> N2 worms grown on control vector bacteria (black; m = 16.9±0.3, n = 81/90) and on <i>cyc-1</i> RNAi bacteria (green; m = 19.6±0.3; n = 85/90; P <i>vs.</i> control <0.0002; percent increase in lifespan: 14). <i>nhr-49</i> mutants grown on control vector bacteria (purple; m = 11.6±0.1; n = 86/89) and on <i>cyc-1</i> RNAi bacteria (red; m = 14.8±0.2; n = 85/95; P <i>vs.</i> control <0.0001; percent increase in lifespan: 22<b>). E</b>–<b>H: NHR-49::GFP expression in adult somatic tissues.</b> NHR-49::GFP is visible in the cytoplasm and nuclei of neurons (E), muscle (F), hypodermis (G) and intestinal cells (H). <i>Pmyo-2::mCherry</i>, the co-injection marker, is seen as red fluorescence in the pharynx in E. I: <b>Rescue of the shortened lifespans of </b><b><i>nhr-49</i></b><b> and </b><b><i>nhr-49</i></b><b>;</b><b><i>glp-1</i></b><b> mutants by the NHR-49::GFP fusion protein.</b> N2 (black; m = 22.8±0.2, n = 81/92), <i>glp-1</i> (green; m = 36.3±0.2, n = 74/104, P <i>vs.</i> N2<0.0001), <i>nhr-49;glp-1</i> (brown; m = 17.9±0.2, n = 104/106, P <i>vs. glp-1</i><0.0001), <i>nhr-49;glp-1;NHR-49::GFP</i> non-transgenic siblings (purple; m = 18.4±0.2, n = 101/107, P <i>vs. glp-1</i><0.0001, P <i>vs. nhr-49</i>;<i>glp-1</i> 0.28), <i>nhr-49;glp-1;NHR-49::GFP</i> (red; m = 35.6±0.4, n = 58/102, P <i>vs. glp-1</i> 0.95, P <i>vs. nhr-49;glp-1</i><0.0001, P <i>vs.</i> non-transgenic siblings <0.0001). All lifespan data are shown as mean lifespan in days (m) ± standard error of the mean (SEM). ‘n’ refers to the number of worms analyzed divided by total number of worms tested in the experiment (some worms were censored from the analysis as described in the <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004829#s4" target="_blank">methods</a> section). P values were calculated using the log rank (Mantel Cox) method. Data from additional trials of these experiments are presented in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004829#pgen.1004829.s014" target="_blank">S2 (panel A)</a>, <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004829#pgen.1004829.s015" target="_blank">S3 (panels B and I)</a>, <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004829#pgen.1004829.s016" target="_blank">S4 (panel C and D) Tables</a>.</p

Integrity of Narrow Epithelial Tubes in the <i>C</i>. <i>elegans</i> Excretory System Requires a Transient Luminal Matrix

<div><p>Most epithelial cells secrete a glycoprotein-rich apical extracellular matrix that can have diverse but still poorly understood roles in development and physiology. Zona Pellucida (ZP) domain glycoproteins are common constituents of these matrices, and their loss in humans is associated with a number of diseases. Understanding of the functions, organization and regulation of apical matrices has been hampered by difficulties in imaging them both <i>in vivo</i> and <i>ex vivo</i>. We identified the PAN-Apple, mucin and ZP domain glycoprotein LET-653 as an early and transient apical matrix component that shapes developing epithelia in <i>C</i>. <i>elegans</i>. LET-653 has modest effects on shaping of the vulva and epidermis, but is essential to prevent lumen fragmentation in the very narrow, unicellular excretory duct tube. We were able to image the transient LET-653 matrix by both live confocal imaging and transmission electron microscopy. Structure/function and fluorescence recovery after photobleaching studies revealed that LET-653 exists in two separate luminal matrix pools, a loose fibrillar matrix in the central core of the lumen, to which it binds dynamically via its PAN domains, and an apical-membrane-associated matrix, to which it binds stably via its ZP domain. The PAN domains are both necessary and sufficient to confer a cyclic pattern of duct lumen localization that precedes each molt, while the ZP domain is required for lumen integrity. Ectopic expression of full-length LET-653, but not the PAN domains alone, could expand lumen diameter in the developing gut tube, where LET-653 is not normally expressed. Together, these data support a model in which the PAN domains regulate the ability of the LET-653 ZP domain to interact with other factors at the apical membrane, and this ZP domain interaction promotes expansion and maintenance of lumen diameter. These data identify a transient apical matrix component present prior to cuticle secretion in <i>C</i>. <i>elegans</i>, demonstrate critical roles for this matrix component in supporting lumen integrity within narrow bore tubes such as those found in the mammalian microvasculature, and reveal functional importance of the evolutionarily conserved ZP domain in this tube protecting activity.</p></div