A Hormonal Signaling Pathway Influencing C. elegans Metabolism, Reproductive Development, and Life Span

AbstractDuring C. elegans development, animals must choose between reproductive growth or dauer diapause in response to sensory cues. Insulin/IGF-I and TGF-β signaling converge on the orphan nuclear receptor daf-12 to mediate this choice. Here we show that daf-9 acts downstream of these inputs but upstream of daf-12. daf-9 and daf-12 mutants have similar larval defects and modulate insulin/IGF-I and gonadal signals that regulate adult life span. daf-9 encodes a cytochrome P450 related to vertebrate steroidogenic hydroxylases, suggesting that it could metabolize a DAF-12 ligand. Sterols may be the daf-9 substrate and daf-12 ligand because cholesterol deprivation phenocopies mutant defects. Sensory neurons, hypodermis, and somatic gonadal cells expressing daf-9 identify potential endocrine tissues. Evidently, lipophilic hormones influence nematode metabolism, diapause, and life span

Dynamic rerouting of the carbohydrate flux is key to counteracting oxidative stress

<p>Abstract</p> <p>Background</p> <p>Eukaryotic cells have evolved various response mechanisms to counteract the deleterious consequences of oxidative stress. Among these processes, metabolic alterations seem to play an important role.</p> <p>Results</p> <p>We recently discovered that yeast cells with reduced activity of the key glycolytic enzyme triosephosphate isomerase exhibit an increased resistance to the thiol-oxidizing reagent diamide. Here we show that this phenotype is conserved in <it>Caenorhabditis elegans </it>and that the underlying mechanism is based on a redirection of the metabolic flux from glycolysis to the pentose phosphate pathway, altering the redox equilibrium of the cytoplasmic NADP(H) pool. Remarkably, another key glycolytic enzyme, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), is known to be inactivated in response to various oxidant treatments, and we show that this provokes a similar redirection of the metabolic flux.</p> <p>Conclusion</p> <p>The naturally occurring inactivation of GAPDH functions as a metabolic switch for rerouting the carbohydrate flux to counteract oxidative stress. As a consequence, altering the homoeostasis of cytoplasmic metabolites is a fundamental mechanism for balancing the redox state of eukaryotic cells under stress conditions.</p

Hormonal Signal Amplification Mediates Environmental Conditions during Development and Controls an Irreversible Commitment to Adulthood

Many animals can choose between different developmental fates to maximize fitness. Despite the complexity of environmental cues and life history, different developmental fates are executed in a robust fashion. The nematode Caenorhabditis elegans serves as a powerful model to examine this phenomenon because it can adopt one of two developmental fates (adulthood or diapause) depending on environmental conditions. The steroid hormone dafachronic acid (DA) directs development to adulthood by regulating the transcriptional activity of the nuclear hormone receptor DAF-12. The known role of DA suggests that it may be the molecular mediator of environmental condition effects on the developmental fate decision, although the mechanism is yet unknown. We used a combination of physiological and molecular biology techniques to demonstrate that commitment to reproductive adult development occurs when DA levels, produced in the neuroendocrine XXX cells, exceed a threshold. Furthermore, imaging and cell ablation experiments demonstrate that the XXX cells act as a source of DA, which, upon commitment to adult development, is amplified and propagated in the epidermis in a DAF-12 dependent manner. This positive feedback loop increases DA levels and drives adult programs in the gonad and epidermis, thus conferring the irreversibility of the decision. We show that the positive feedback loop canalizes development by ensuring that sufficient amounts of DA are dispersed throughout the body and serves as a robust fate-locking mechanism to enforce an organism-wide binary decision, despite noisy and complex environmental cues. These mechanisms are not only relevant to C. elegans but may be extended to other hormonal-based decision-making mechanisms in insects and mammals

High amounts of DA are required for normal adult development.

<p>(A) Images of dauer, arrested L3, abnormal development Mig and Cut worms, and normal adults. Yellow hatched area encloses the gonad, v, vulva. (B) Distribution of developmental stages as a function of DA, scored 48 hph. Means of dauer (red), abnormal development (arrested L3, Mig and Cut; yellow) and normal adult (L3, L4, and young adult; blue) phenotype in <i>daf-9(dh6)</i> worms. (C) Distribution of developmental stages in the <i>daf-9(rh50)</i> background. (D) Distribution of stages in the adult fraction of phenotypes. Means of population proportions of stages indicate the relative developmental rate at each concentration of DA scored at 48 hph. Error bars represent means ± standard deviations across three biological replicates, <i>N</i>>500. Mig, gonad migratory defective; Cut, cuticle defective; YA; young adult. * Worms were gravid the next day.</p

Timing requirements of Δ7-DA.

<p>Dauer, abnormal development and normal adult fates as a function of exposure times to 100 nM Δ7-DA. (A) <i>daf-9(dh6)</i> worms start responding to Δ7-DA at 15 hph and require an additional 12 h of Δ7-DA for normal adult development. Top, representative colored bars indicating the shift experiment: red bars indicate EtOH carrier and blue bars indicate Δ7-DA. Bottom, normal adult (blue), abnormal adult (yellow), and dauer (red) bars indicate the population fraction per time point. (B) Worms become refractory to Δ7-DA at 33 hph, the same time that they commit to dauer. N2 indicates worms shifted from unfavorable to favorable conditions as indicated in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001306#pbio-1001306-g001" target="_blank">Figure 1C</a>, and points indicate dauer proportions (abnormal development is considered non-dauer in this panel). (C) Pulses of Δ7-DA indicate the minimal time necessary for normal development when added at 15 hph. Top, length of pulses. Bottom, normal adult (blue), abnormal development (yellow), and dauer (red) bars indicate the population fraction per time point. (D) Worms have no memory of previous exposure to Δ7-DA before the L1/L2 molt. Pie charts indicate proportions of dauers (red), abnormal development (yellow), and normal adults (blue) as a function of total amount of time exposed to Δ7-DA (<i>x</i>-axis) when exposed to Δ7-DA at different hours post-hatch (<i>y</i>-axis). <i>N</i>>100 for all time points.</p

Commitment to dauer or reproductive development.

<p>(A) Developmental molt times of <i>C. elegans</i> N2 strain growing in favorable (blue) or unfavorable (red) conditions. (B–E) Time courses of commitment as a function of environmental conditions (pheromone). Top, representative colored bars indicate shifts to unfavorable conditions (red) or favorable conditions (blue). Bottom, means of dauer frequencies between biological replicates ± standard deviation. Numbers in parentheses indicate total worms per time point. (B) Period of pheromone sensitivity during L1 and L2: worms respond to pheromone between 12 and 18 hph. (C) Point of commitment to dauer: worms commit to dauer 33 hph denoted by the red dashed gridline. (D) Point of commitment from L2d to L3: worms commit to L3 after a 3 h pulse in favorable conditions when shifted at 24 hph. (E) Start time of pulse shifts to favorable conditions during L2d. Pheromone was added to worms 3 h post-shift to favorable conditions. Cultures shifted to favorable conditions at 33 hph show a higher ratio of dauers since worms commit to dauer at 33 hph (red dashed gridline). Control, worms grown without pheromone. Dauer, worms grown in 3% (v/v) pheromone with no shifts.</p

Dauer pheromone regulates the threshold for reproductive development.

<p>(A–C) Distribution of the phenotypes of the strain <i>daf-9(dh6)</i> when supplemented with DA: dauers (A), abnormal adults (B), and normal adults (C), as a function of DA and pheromone. Each pixel on the heat map is the mean fraction of population (see bar on the right for quantification) developing in the specific category, <i>N</i>>300 per pixel. Partition of abnormal adults and adults into sub-categories is detailed in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001306#pbio.1001306.s003" target="_blank">Figure S3</a>. (D) Concentrations of DA required to surpass the dauer bypass DA threshold as a function of pheromone. Yellow dashed line indicates 90% non-dauers in the population. (E) Concentrations of DA required for normal adult development without any arrested L3, Mig, or Cut phenotypes. Blue dashed line indicates 90% normal adults in the population. (F) Regression analysis of panels D and E; points correspond to 90% non-dauer (Yellow, 3D) and 90% normal adult (Blue, 3E, see Experimental Procedures for details of regression). Normal adult development requires an additional 30 nM DA above the amount for dauer bypass. Mig, gonad migratory defective; YA, young adult.</p

Hypodermal <i>daf-9</i> expression propagates from anterior to posterior upon commitment to the L3 fate.

<p>(A, top) Fluorescent images of worms at each time point are shown at shift from unfavorable to favorable at 0 (24 hph) h (leftmost image) through 12 hph (rightmost image). Arrowheads mark the XXX cells. (A, bottom) Expression of hypodermal <i>daf-9</i> was quantified along the anterior posterior axis in 4–6 worms in each time point. Each green shaded histogram represents the mean grey value of DAF-9::GFP per worm, normalized to length. Different worms were imaged at each time point (see <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001306#pbio.1001306.s009" target="_blank">Text S1</a> for details of analysis). (B–D) XXX cells and Δ7-DA are required to initiate hypodermal daf-9 expression and reproductive development. (B) Cells were ablated during L2d and recovered in favorable conditions. (C) L2d ablated worms were let to recover on 1, 5, or 10 nM Δ7-DA. All worms expressing hypodermal <i>daf-9</i> developed into normal adults with no Mig or Cut phenotypes. (D) Worms were grown to L2d, XXX cells were ablated after commitment to L3 at 27 hph. * <i>p</i><1×10⁻⁴, ** <i>p</i><1×10⁻¹⁰.</p

A feedback loop amplifies a DA signal leading to coordinate development.

<p>(A) Environmental conditions overlap with DA time of action. Top, growth in favorable conditions (blue) during the induction period commits worms to adulthood. Committed worms develop into adults even if shifted to unfavorable conditions (red). Commitment to adulthood correlates with the time of action of DA. Middle and bottom: worms grown in unfavorable conditions during the induction period develop into L2d worms which decide between regular development and alternative development during the integration period (between 15 and 33 hph, hashed grey lines). Worms commit to the dauer fate at 33 hph or to adult development if exposed to favorable conditions for 3 h. Development to dauer correlates with no DA production, and development to adulthood correlates with production of DA. (B) Noisy and uncertain environmental information is measured by sensory neurons and reduced in complexity into the four signaling pathways. Information complexity is reduced further to the XXX cells, the primary source of DA. If nascent amounts of DA produced by the XXX cells bypass the dauer DA threshold, worms will develop into reproductive adults, and if DA levels are under the threshold, worms will develop into dauers. Upon commitment to reproductive development, DA originating from the XXX cells will initiate the hypodermal <i>daf-9</i> positive feedback loop, thus increasing the amounts of DA and thus locking the adult decision and producing sufficient amounts of DA for complete adult development. The positive feedback loop canalizes development guaranteeing that sufficient amounts of DA are produced so that abnormal phenotypes are not expressed in adult worms.</p

NFYB-1 regulates mitochondrial function and longevity via lysosomal prosaposin

Mitochondria are multidimensional organelles whose activities are essential to cellular vitality and organismal longevity, yet underlying regulatory mechanisms spanning these different levels of organization remain elusive(1-5). Here we show that Caenorhabditis elegans nuclear transcription factor Y, beta subunit (NFYB-1), a subunit of the NF-Y transcriptional complex(6-8), is a crucial regulator of mitochondrial function. Identified in RNA interference (RNAi) screens, NFYB-1 loss leads to perturbed mitochondrial gene expression, reduced oxygen consumption, mitochondrial fragmentation, disruption of mitochondrial stress pathways, decreased mitochondrial cardiolipin levels and abolition of organismal longevity triggered by mitochondrial impairment. Multi-omics analysis reveals that NFYB-1 is a potent repressor of lysosomal prosaposin, a regulator of glycosphingolipid metabolism. Limiting prosaposin expression unexpectedly restores cardiolipin production, mitochondrial function and longevity in the nfyb-1 background. Similarly, cardiolipin supplementation rescues nfyb-1 phenotypes. These findings suggest that the NFYB-1-prosaposin axis coordinates lysosomal to mitochondria signalling via lipid pools to enhance cellular mitochondrial function and organismal health