Natural Variation, Development, and Adaptive Phenotypes in C. elegans

Natural Variation, Development, and Adaptive Phenotypes in C. elegans Bradly Alicea http://publish.illinois.edu/bradly-alicea Fly/Worm Club, UIUC, April 22, 2015 with input from Nathan Schroeder, Rebecca Androwski, Kristin Flatt

H: C. elegans exhibits polyphenic states and related plastic responses related to environmental challenges. Polyphenism: alternative phenotypes (with a parallel life-history) produced by the same genotypic background. * alternative phenotypes may be due to genes selected for specialized adaptive function (e.g. J Evol Biol, 22(3), 599-611 (2009). * related adaptive plastic responses (e.g. quiescence) can also occur due to physiological constraints across lifehistory (Cell Cycle, 11(9), 1672-1679 -- 2012). Environmental Trigger Polyphenic State, Adaptive Plasticity Normal phenotype Dauer phenotype Adaptive Genotype (e.g. daf-c) Associated variants that modify genotypic response to environment

Mosaic (Determinant) Built-in Instructions Two Types of Development Regulative (Indeterminant) Instructions from outside Independence from environment Common in Protostomes "Declarative" process (e.g. go 10ft, turn left) Material arranged asymmetrically in egg (e.g. cytoplasmic determinants) Gradients of mrna or protein in egg Blastomeres have no totipotent potential Dependence on environment (conditional) Common in Deuterostomes "Approximate" process (e.g. (go until condition x, turn left) Signaling mechanisms (e.g. multiple pathways) Close cell contacts (e.g. paracrine, juxtacrine signaling) Blastomeres have totipotent potential

What s so interesting about mosaic development? Results in a lineage tree that is largely invariant from one individual to another. All cells have clear mother-daughter relationships, and sublineages consist of cells with a common ancestor. * sublineages (like morphogen gradients) usually contribute to the formation of a single organ (spatially restricted). Joshi, Mani, Rothman Cellular organization in C.elegans embryo. Morpho13 Wiki.

What s so interesting about mosaic development? Results in a lineage tree that is largely invariant from one individual to another. All cells have clear mother-daughter relationships, and sublineages consist of cells with a common ancestor. * sublineages (like morphogen gradients) usually contribute to the formation of a single organ (spatially restricted). * Labile fate restricted to Germline Stem Cells (GSCs). Joshi, Mani, Rothman Cellular organization in C.elegans embryo. Morpho13 Wiki. Kimble, Seidel C. elegans germline stem cells and their niche. StemBook, 1.95.1 (2013)

Polyphenism in a Mosaic Organism (C. elegans): dauer phenotype Alternative larval stage (dauer) occurs as L1 larvae experience environmental stress, could be viewed as archetypical C. elegans polyphenism (Q. Rev Biol, 88(3), 185-218 2013). * dauer larval stage triggered by environmental cues (temperature, starvation) and mitigated by genotype (daf-c background, specific variants). Dauer stage involves an initial entry stage, the state of diapause itself, and a post-diapasual recovery stage. * dauer stage can extend the lifespan of the organism by weeks (due to suspended animation nature of diapausal stage). One interesting source of developmental variation is in the AMPK pathway: * part of the IIS/TOR pathway, contributes to caste differentiation in social insects and organ size regulation in Drosophila. References: Frontiers Ecol Evol, 4, 263 (2013). IUBMB Life, 61(6), 607-612 (2012).

Polyphenism in a Mosaic Organism (C. elegans): dauer phenotype Polyphenism: two/more distinct phenotypes produced by same genotype. * dauer larval stage triggered by environmental cues (temperature, starvation) and mitigated by genotype (daf-c background, specific variants). Two observations suggest that while dauers exhibit the features of a polyphenic state, a broader set of adaptive mechanisms may be at play: 1) Up to 70% of dauers are abnormal in some way (alternate phenotype with variation). 2) Conventional methods of generating dauer worms (detergents, florescent beads) bias the surviving worms towards certain trait configurations. Are some worms predisposed to survival over others? Are there partial dauer stages in which some survivors exhibit a subset of typical dauer traits?

Different genotypes yield different rates of survival (conversion of phenotype to Dauer stage) Genotype Lifespan (days @ 25C) * n WT (control) 29.6 230 aak-1 (tm1944) 15 80 aak-2 (ok524) 8.1 200 aak-2 (rr48) 10.5 108 aak-1; aak-2 7.5 213 Genotype Percent Survival ** n daf-7 (e1372); lag-2 (q420) glp-1 (e1372); daf-7 (e1372) glp-1 (q231); daf-7 (e1372) * Nature, 457, 210-214 (2009). ** Development, 135, 2538-2592 (2008). 81.3 200 84.7 354 37.7 450 AMPK (aak) mutants: lipid metabolism defects (e.g. rapid consumption of energy) * energetic stress = vital organ failure in general, lack of osmoregulation during dauer. * aak-2 is not required for increased survival in daf-2 mutants, but aak-2 mutants show decreased ability to survive harsh conditions. * daf-7 is a temperature-dependent switching mechanism (25C = dauer on ). * lag-2 and glp-1 implicated in neuronal NOTCH signaling cascade, maintenance of dauer phenotype.

EPISTATIC MODEL, HETEROCHRONIC GENES: Morphogenetic Pathway: control hypodermal cell differentiation, transition into adulthood. Temporal Pathway: triggers timing of entry into Dauer. lin-28 lin-29 lin-4 lin-14 Genes and Development 3, 2039-2049 (1989). Part of a linear epistasis pathway, group of 23 genes that affect dauer formation (Genetics, 130, 105-123, 1992). * mutations in lin-4, lin-14, lin-28, lin-29 result in precocious or retarded phenotypes (changes in timing). As a polyphenism, dauer transformation occurs in a switch-like fashion, buffering the organism from harsh environmental conditions. * there may also be adaptive states (reaction norms) related to this polyphenism that use parts of the more formal polyphenic response in responding to harsh environments (partial dauer). * does not result in a parallel mode of life-history, but is a way for an organism to explore its adaptive potential (Evol Devel, 5(1), 9-18 2003).

Short-term Experimental Evolution: selection for adaptive plasticity Diaz and Viney (Ecology and Evolution, 5(6), 1343 2015): 5 generations of artificial selection applied to C. elegans populations. * select for worms that form dauer phenotypes (allow surviving worms to recover and reproduce). Dauer Pheromone (+) Dauer Pheromone (-) HI food concentration HI food, Pheromone + HI food, Pheromone - LO food concentration LO food, Pheromone + LO food, Pheromone - Ascaroside = dauer-specific pheromone (chemosensory signal). Food scarcity = metabolic signal. Outcomes: * pheromone acts as positive selection. * HI food, pheromone + condition selects for life-history (dauer) plasticity. * adaptation is related to reproductive timing. * diversity of plastic responses result from selection being imposed.

Populations that exhibit halting behavior: * observed in single, double AMPK mutant phenotypes. Less often observed in non-ampk mutant phenotypes. * observations of mass movement arrest in a densely or sparsely populated plate. Observe Halting Behavior (probabilistic recovery) Needle Phenotypes Frozen Phenotypes Long, thin, and found in bundles. Occasional individuals found slowly gliding through bundle. Curved, semi-coiled found singularly. Occasional slow-moving individuals with slight exploratory behavior.

Needle Phenotype: long, thin, rigid phenotype. Sometimes found in bundles, individuals can slowly slide out of bundles. * can be spontaneous (in damp-ish OP50 medium) or induced (SDS 1%). * survival from this state is probabilistic some of these worm bodies are dead, some of them will recover. GFP + aak-1;aak-2 (after SDS treatment) aak-1; aak-2 isolate from needles (1d)

Frozen Phenotype: less rigid phenotypes that do not move or move very slowly. Spontaneous state that occurs across an entire population (plate) regardless of starvation or crowding. Often occurs in plates that accrue sticky residue (pheromone?). aak-1 aak-1; aak-2 Potentially viable phenotypes; will recover after 24-36h, or sooner as isolate

Frozen Phenotype: less rigid phenotypes that do not move or move very slowly. Spontaneous state that occurs across an entire population (plate) regardless of starvation or crowding. aak-1 aak-1; aak-2 Dead Worms Recoverable Worms Also observed in non-aak phenotypes (data not shown): frozen phenotypes express GFP and create viable isolates as in normal phenotypes, but original culture does not recover.

Populations that exhibit halting behavior: * observed in single, double AMPK mutant phenotypes. Less often observed in non-ampk mutant phenotypes. * observations of mass movement arrest in a densely or sparsely populated plate. Bias in non-aak-1/2 phenotypes: * aak-1/2 mutant genotype -- lower chance of survival (environmental selection). * wt background -- higher chance of survival (environmental selection). * aak-1/2 + compensatory mechanism -- higher chance of survival (buffering of the aak-1/2 phenotype)? When you expose worms to starvation conditions or large amounts of stress, do you inadvertently select for background alleles (compensatory variants)? Does pushing worms into Dauer select against aak-1/2 phenotype in a population? Different ways to get to the adaptive phenotype (e.g. starvation vs. SDS vs. temperature)?

Final Word: populations are made up of individuals! Needle and Frozen physiological responses to environmental stress as adaptive phenotypes: * how are these responses related to developmental and genetic mechanisms? * what percentage of individuals survive adverse environmental conditions? Interactions between genetic background and what phenotypic adaptations they exhibit. Taking this further: need to examine individuality or intra-population diversity. * isolate and count survivors, distinguish from non-survivors. Characterize effects of selection on various genetic strains. * using dauer stage (polyphenism) as a template, how many dauer-like traits are exhibited during adaptive plasticity (e.g. amongst surviving phenotypes)? * what dauer-like traits are absent from surviving phenotypes? * are there uncharacterized mutations in the background that enhance the ability of some genotypes to survive better than others?