Browsing by Author "Baugh, L Ryan"

Nearly all animals live in environments with fluctuations in nutrient availability. The ability to sense and respond to these changes is essential for survival. Nutrition impacts physiology immediately, but can also have long-lasting effects across generations. The nematode Caenorhabditis elegans is particularly well-adapted to thrive in conditions of variable food availability. Here we find that starvation responses in C. elegans are largely independent of the larval stage at which worms experience starvation. Starvation in worms results in shrinkage, delayed growth upon recovery, and ultimately death. In order to adapt to starvation, metabolism is dramatically altered. At a gross level, this can be seen in a reduction of mitochondrial genomes and a more fragmented network of mitochondria.

Insulin-like signaling is a key cell signaling pathway controlling nutrient responses. We interrogate the role of insulin-like signaling in regulation of the acute starvation response. We show that daf-16/FoxO restructures carbohydrate metabolism by driving carbon flux through the glyoxylate shunt and gluconeogenesis and into synthesis of trehalose, a disaccharide of glucose. Trehalose is a well-known stress protectant, capable of preserving membrane organization and protein structure during abiotic stress. Metabolomic, genetic, and pharmacological analyses confirm increased trehalose synthesis and further show that trehalose not only supports survival as a stress protectant, but also serves as a glycolytic input. Further, we provide evidence that metabolic cycling between trehalose and glucose is necessary for this dual function of trehalose. This work demonstrates that daf-16/FoxO promotes starvation resistance by shifting carbon metabolism to drive trehalose synthesis, which in turn supports survival by providing an energy source and acting as a stress protectant.

In addition to acute changes in response to the nutrient environment, effects can persist intergenerationally. Maternal effects of environmental conditions produce intergenerational phenotypic plasticity. Adaptive value of these effects depends on appropriate anticipation of environmental conditions in the next generation, and mismatch between conditions may contribute to disease. However, regulation of intergenerational plasticity is poorly understood. Dietary restriction (DR) delays aging but maternal effects have not been investigated. We demonstrate maternal effects of DR in the roundworm C. elegans. Worms cultured in DR produce fewer but larger progeny. Nutrient availability is assessed in late larvae and young adults, rather than affecting a set point in young larvae, and maternal age independently affects progeny size. Reduced signaling through the insulin-like receptor daf-2/InsR in the maternal soma causes constitutively large progeny, and its effector daf-16/FoxO is required for this effect. nhr-49/Hnf4, pha-4/FoxA, and skn-1/Nrf also regulate progeny-size plasticity. Genetic analysis suggests that insulin-like signaling controls progeny size in part through regulation of nhr-49/Hnf4, and that pha-4/FoxA and skn-1/Nrf function in parallel to insulin-like signaling and nhr-49/Hnf4. Furthermore, progeny of DR worms are buffered from adverse consequences of early-larval starvation, growing faster and producing more off- spring than progeny of worms fed ad libitum. These results suggest a fitness advantage when mothers and their progeny experience nutrient stress, compared to an environmental mismatch where only progeny are stressed. This work reveals maternal provisioning as an organismal response to DR, demonstrates potentially adaptive intergenerational phenotypic plasticity, and identifies conserved pathways mediating these effects.

The roundworm C. elegans reversibly arrests larval development during starvation, but extended early-life starvation reduces reproductive success. Maternal dietary restriction (DR) buffers progeny from starvation as young larvae, preserving reproductive success. However, the developmental basis of reduced fertility following early-life starvation is unknown, and it is unclear how maternal diet modifies developmental physiology in progeny. We show here that extended starvation in first-stage (L1) larvae followed by unrestricted feeding results in a variety of developmental abnormalities in the reproductive system, including proliferative germ-cell tumors and uterine masses that express neuronal and epidermal cell-fate markers. We found that maternal DR and reduced maternal insulin/IGF signaling (IIS) increase oocyte provisioning of vitellogenin lipoprotein, reducing penetrance of starvation-induced abnormalities in progeny, including tumors. Furthermore, we show that maternal DR and reduced maternal IIS reduce IIS in progeny. daf-16/FoxO and skn-1/Nrf, transcriptional effectors of IIS, are required in progeny for maternal DR and increased vitellogenin provisioning to suppress starvation-induced abnormalities. daf-16/FoxO activity in somatic tissues is sufficient to suppress starvation-induced abnormalities, suggesting cell-nonautonomous regulation of reproductive system development. This work reveals that early-life starvation compromises reproductive development and that vitellogenin-mediated intergenerational insulin/IGF-to-insulin/IGF signaling mediates adaptation to nutrient availability. Using our SIA model, we go on to show that early-life starvation persistently activates PQM-1/SALL2 with pervasive effects on adult gene expression, including prominent effects on membrane biology. Early-life starvation increases fatty-acid synthetase fasn-1/FASN expression in pqm-1/SALL2-dependent fashion, and both genes promote SIA. Lipidomic analysis implicates phosphatidylcholine, and unsaturated phosphatidylcholine supplementation suppresses SIA. The fatty-acid desaturases fat-1 and fat-4 inhibit and promote SIA, respectively, revealing a role of arachidonic acid-containing phosphatidylcholine, the Lands cycle, and eicosanoid signaling. Indeed, fat-4 increases eicosanoid levels in adults subjected to early-life starvation, and N-acetylcysteine treatment suppresses SIA. This work shows that early-life starvation and IIS converge on PQM-1/SALL2 to affect adult lipid metabolism and eicosanoid signaling, affecting stem-cell proliferation and tumor formation.

Developmental physiology is very sensitive to nutrient availability. For instance, in the nematode C. elegans, newly hatched L1-stage larvae require food to initiate postembryonic development. Despite the essential role of food in C. elegans development, the contribution of food perception versus ingestion on physiology has not been delineated. We used a pharmacological approach to uncouple the effects of food (bacteria) perception and ingestion in C. elegans. Perception was not sufficient to promote postembryonic development in L1-stage larvae. However, L1 larvae exposed to food without ingestion failed to develop upon return to normal culture conditions, instead displaying an irreversible arrest phenotype. Inhibition of gene expression during perception rescued subsequent development, demonstrating that the response to perception without feeding is deleterious. Perception altered DAF-16/FOXO subcellular localization, reflecting activation of insulin/IGF signaling (IIS). However, genetic manipulation of IIS did not modify the irreversible arrest phenotype caused by food perception, revealing that wild-type function of the IIS pathway is not required to produce this phenotype and that other pathways affected by perception of food in the absence of its ingestion are likely to be involved. Gene expression and Nile red staining showed that food perception could alter lipid metabolism and storage. We found that starved larvae sense environmental polypeptides, with similar molecular and developmental effects as perception of bacteria. We conclude that actual ingestion of food is required to initiate postembryonic development in C. elegans. We also conclude that polypeptides are perceived as a food-associated cue in this and likely other animals, initiating a signaling and gene regulatory cascade that alters metabolism in anticipation of feeding and development, but that this response is detrimental if feeding does not occur.

The C. elegans insulin-like signaling network supports homeostasis and developmental plasticity. The genome encodes 40 insulin-like peptides and one known receptor. Feedback regulation has been reported, but the extent of feedback and its effect on signaling dynamics in response to changes in nutrient availability has not been determined. We measured mRNA expression for each insulin-like peptide, the receptor daf-2, components of the PI3K pathway, and its transcriptional effectors daf-16/FOXO and skn-1/Nrf at high temporal resolution during transition from a starved, quiescent state to a fed, growing state in wild type and mutants affecting daf-2/InsR and daf-16/FOXO. We also analyzed the effect of temperature on insulin-like gene expression. We found that most PI3K pathway components and insulin-like peptides are affected by signaling activity, revealing pervasive positive and negative feedback regulation at intra- and inter-cellular levels. Reporter gene analysis demonstrated that the daf-2/InsR agonist daf-28 positively regulates its own transcription and that the putative agonist ins-6 cross-regulates DAF-28 protein expression through feedback. Our results show that positive and negative feedback regulation of insulin-like signaling is widespread, giving rise to an organismal FOXO-to-FOXO signaling network that supports homeostasis during fluctuations in nutrient availability.

L1 arrest (or "L1 diapause") is associated with increased stress resistance, supporting starvation survival. Loss of the transcription factor daf-16/FOXO results in arrest-defective and starvation-sensitive phenotypes. We show that daf-16/FOXO regulates L1 arrest cell-nonautonomously, suggesting that insulin/IGF signaling regulates at least one additional signaling pathway. We used mRNA-seq to identify candidate signaling molecules affected by daf-16/FOXO during L1 arrest. dbl-1/TGF-β, a ligand for the Sma/Mab pathway, daf-12/NHR, and daf-36/oxygenase, an upstream component of the daf-12 steroid hormone signaling pathway, were up-regulated during L1 arrest in a daf-16/FOXO mutant. Using genetic epistasis analysis, we show that dbl-1/TGF-β and daf-12/NHR steroid hormone signaling pathways are required for the daf-16/FOXO arrest-defective phenotype, suggesting that daf-16/FOXO represses dbl-1/TGF-β, daf-12/NHR and daf-36/oxygenase. The dbl-1/TGF-β and daf-12/NHR pathways have not previously been shown to affect L1 development, but we found that disruption of these pathways delayed L1 development in fed larvae, consistent with these pathways promoting development in starved daf-16/FOXO mutants. Though the dbl-1/TGF-β and daf-12/NHR pathways are epistatic to daf-16/FOXO for the arrest-defective phenotype, disruption of these pathways does not suppress starvation sensitivity of daf-16/FOXO mutants. This observation uncouples starvation survival from developmental arrest, indicating that DAF-16/FOXO targets distinct effectors for each phenotype and revealing that inappropriate development during starvation does not cause the early demise of daf-16/FOXO mutants. We show that daf-16/FOXO promotes developmental arrest cell-nonautonomously by repressing pathways that promote larval development.

Bet-hedging strategies are used by organisms to survive in

unpredictable environments. To pursue a bet-hedging strategy, an

organism must produce multiple phenotypes from a single genotype. What

molecular mechanisms allow this to happen? To address this question, I

created a synthetic system that displays bet-hedging behavior, and

developed a new technique called `TrackScar' to measure the fitness

and stress-resistance of individual cells. I found that bet-hedging

can be generated by actively sensing the environment, and that

bet-hedging strategies based on active sensing need not be

metabolically costly. These results suggest that to understand how

bet-hedging strategies are produced, microorganisms must be

examined in the actual environments that they come from.

Fluctuations in nutrient availability occur for nearly all species, and adaptation to endure starvation conditions is essential. Genetic pathways involved in regulating starvation resistance are implicated in aging and complex diseases such as cancer, diabetes, and obesity in humans. Consequences of experiencing starvation persist later in life and subsequent generations, suggesting epigenetic regulation. However, much is still unknown about how starvation resistance is regulated and the contributions of different types of regulation. The roundworm Caenorhabditis elegans reversibly arrests development in the absence of food and can endure starvation for several weeks. Here, we investigate how transcriptional, epigenetic, and genetic regulation impact starvation resistance during developmental arrest in C. elegans. Gene expression dynamics change quickly during the first few hours of starvation, and broadly conserved transcription factors are required for starvation survival. However, temporal- and tissue-specific requirements of transcription for supporting starvation survival and recovery are largely unknown. In chapter 2, we used mRNA-seq combined with temporal degradation of RNA Polymerase II in the soma and germline to better understand gene regulation throughout arrest. We find that transcription is required in the soma for survival early in starvation but is dispensable thereafter, and known transcriptional regulators primarily act early in arrest. In contrast, the germline is transcriptionally quiescent throughout starvation, but germline transcripts are relatively stable compared to somatic transcripts. This reveals alternative gene-regulatory strategies in the soma and germline during starvation-induced developmental arrest, with the soma relying on a robust early transcriptional response while the germline relies on mRNA stability to maintain integrity. Phenotypic plasticity is facilitated by epigenetic regulation, and remnants of such regulation may persist after plasticity-inducing cues are gone, even affecting germ cells to impact subsequent generations. However, the relationship between plasticity and transgenerational epigenetic memory is not understood. Dauer diapause provides an opportunity to determine how a plastic response to the early-life environment affects traits later in life and in subsequent generations. In chapter 3, we find that, after extended diapause, postdauer worms initially exhibit reduced reproductive success and greater interindividual variation. In contrast, F3 progeny of postdauers display increased starvation resistance and lifespan, revealing potentially adaptive transgenerational effects. Transgenerational effects are dependent on the duration of diapause, indicating an effect of extended starvation. In agreement, RNA-seq demonstrates a transgenerational effect on nutrient-responsive genes. This work reveals complex effects of nutrient stress over different time scales in an animal that evolved to thrive in feast and famine. Many conserved genes and pathways regulate starvation resistance, but most genetic analysis in C. elegans has been restricted to a single genetic background, potentially restricting identification of additional genes. Hundreds of genetically distinct wild strains of C. elegans have been whole-genome sequenced and can be used for GWAS. In chapters 4 and 5, we implemented two high-throughput sequencing approaches, RAD-seq and MIP-seq, to determine relative starvation resistance of over 100 wild strains over time. We used GWAS to identify QTL associated with starvation resistance, near-isogenic lines to validate QTL, and CRISPR gene editing to modify specific genes within QTL. We focused on genes in the insulin receptor-like domain (irld) family, as this family has been virtually uncharacterized, but the genes share homology with the sole known insulin-like receptor in C. elegans, DAF-2, which is a major regulator of starvation resistance. We found that specific variants in two members of the irld family confers increased starvation resistance in multiple genetic backgrounds, and this is dependent on the transcription factor downstream of insulin signaling, DAF-16/FOXO. Thus, this work shows that natural genetic variation in novel modifiers of insulin-signaling regulates starvation resistance.

Animals must coordinate development with fluctuating nutrient availability. Nutrient availability governs post-embryonic development in Caenorhabditis elegans: larvae that hatch in the absence of food do not initiate post-embryonic development but enter "L1 arrest" (or "L1 diapause") and can survive starvation for weeks, while rapidly resume normal development once get fed. Insulin-like signaling (IIS) has been shown to be a key regulator of L1 arrest and recovery. However, the C. elegans genome encodes 40 insulin-like peptides (ILPs), and it is unknown which peptides participate in nutritional control of L1 arrest and recovery. Work in other contexts has identified putative receptor agonists and antagonists, but the extent of specificity versus redundancy is unclear beyond this distinction.

We measured mRNA expression dynamics with high temporal resolution for all 40 insulin-like genes during entry into and recovery from L1 arrest. Nutrient availability influences expression of the majority of insulin-like genes, with variable dynamics suggesting complex regulation. We identified 13 candidate agonists and 8 candidate antagonists based on expression in response to nutrient availability. We selected ten candidate agonists (daf-28, ins-3, ins-4, ins-5, ins-6, ins-7, ins-9, ins-26, ins-33 and ins-35) for further characterization in L1 stage larvae. We used destabilized reporter genes to determine spatial expression patterns. Expression of candidate agonists was largely overlapping in L1 stage larvae, suggesting a role of the intestine, chemosensory neurons ASI and ASJ, and the interneuron PVT in systemic control of L1 development. Transcriptional regulation of candidate agonists was most significant in the intestine, as if nutrient uptake was a more important influence on transcription than sensory perception. Scanning in the 5' upstream promoter region of these 40 ILPs, We found that transcription factor PQM-1 and GATA putative binding sites are depleted in the promoter region of antagonists. A novel motif was also found to be over-represented in ILPs.

Phenotypic analysis of single and compound deletion mutants did not reveal effects on L1 recovery/developmental dynamics, though simultaneous disruption of ins-4 and daf-28 extended survival of L1 arrest without enhancing thermal tolerance, while overexpression of ins-4, ins-6 or daf-28 shortened L1 survival. Simultaneous disruption of several ILPs showed a temperature independent, transient dauer phenotype. These results revealed the relative redundancy and specificity among agonistic ILPs.

TGF- β and steroid hormone (SH) signaling have been reported to control the dauer formation along with IIS. Our preliminary results suggest they may also mediate the IIS control of L1 arrest and recovery, as the expression of several key components of TGF-β and SH signaling pathway genes are negatively regulated by DAF-16, and loss-of-function of these genes partially represses daf-16 null phenotype in L1 arrest, and causes a retardation in L1 development.

In summary, my dissertation study focused on the IIS, characterized the dynamics and sites of ILPs expression in response to nutrient availability, revealed the function of specific agonistic ILPs in L1 arrest, and suggested potential cross-regulation among IIS, TGF-β signaling and SH signaling in controlling L1 arrest and recovery. These findings provide insights into how post-embryonic development is governed by insulin-like signaling and nutrient availability.

Starvation during early human development produces epigenetic effects that could be adaptive if famine persists. We modeled the response to early starvation exposure in C. elegans using `L1 arrest,' a reversible developmental arrest in the first larval stage brought on by hatching in the absence of food. We found lifelong developmental effects following recovery from extended L1 arrest. Remarkably, some epigenetic effects persist for multiple generations. After extended starvation, development is delayed, producing smaller adults, fertility is reduced, and stress resistance increases. Starvation causes a striking amount of phenotypic variation among isogenic individuals, and those that develop slowest are least fertile and most stress resistant. However, increased stress resistance appears to be characteristic of recovering animals following any amount of L1 arrest but decreasing as the animal grows, possibly by size-dependent dilution. We assessed starved animals for signs of autophagy-related feeding defects and found low rates of pumping and feeding as well as grinding defects. A retrospective pumping assay revealed that after extended starvation, animals with lower pumping rates at the L1 stage tended to grow slower. Our work shows that environmental conditions and life history have transgenerational life history effects on several organismal traits, and that these traits appear to be rooted in nutrient-deprivation secondary to autophagy-related feeding defects. However, the manner by which these effects are transmitted transgenerationally remains an open and interesting question.

The advent of high coverage and low cost sequencing technologies has allowed for

newer and more powerful approaches in molecular and population genetics. Transposon

sequencing, where genome-saturated mutant populations allele frequencies are

measured before and after selection, functionally characterizes each and every gene

in the genome in a single experiment. The approach has been successfully applied

to a variety of phenotypes in a variety of unicellular systems: growth and motility

in E. coli, synthetic genetic interactions in yeast, and in vitro pathogen-resistance in

mammalian cell lines. However, transposon insertion typically produces null alleles,

which can be valuable to identify gene function, but evolutionary insight relies on

identifcation of naturally occurring polymorphisms affecting the trait of interest.

Genome-wide association studies (GWAS) can be used to study the effect of natural

genetic variation on a trait, but they grow prohibitively expensive if the number of

individuals to genotype and phenotype becomes large.

Here I describe the application of transposon sequencing and pooled sequencing

GWAS in the whole metazoan model, Caenorhabditis elegans. Transposon sequencing

has not been previously implemented in an animal model. I have sequenced a control

library using our method, C. elegans transposon sequencing (CeTnSeq). We have

constructed a new Mos1 transposon mutator strain that is more convenient to use

than the existing strain and allows for extra-chromosomal insertions to be degraded

by restriction digest. My preliminary results show that our method is qualitatively

effective at identifying transposon insertion sites, but suffers from PCR duplication

error. I propose to optimize the number of PCR cycles in the library and to include

unique molecular identifiers (UMI) in the library adaptor. I also show that the

restriction digest is effective at removing extra-chromosomal array insertions from

the library.

I constructed simulation models to help design optimal Ce-TnSeq experiments

with respect to statistical power for a proposed starvation survival assay. I considered

many parameters affecting the design, including: culture size, number of generations,

expected effect size, sequencing coverage, and sample size. I show that the number

of homozygous mutant animals in the screen is a critical factor in the design of

experiments. I also saw diminishing returns with respect to increasing sample size

and sequencing depth. These simulations will be invaluable in designing future Ce-

TnSeq experiments and identifying critical aspects of the protocol to optimize.

We performed pooled sequencing (using restriction-site associated DNA sequencing)

on a population of 95 wild isolates subjected to starvation. I identified strains

that were resistant and sensitive to starvation, and we verified these results using

traditional methods. We used our population sequencing data to perform an association

study of starvation survival across the 95 strains, and identified two statistically

significant quantitative trait loci.