[
Aging, Metabolism, Stress, Pathogenesis, and Small RNAs, Madison, WI,
2010]
In Caenorhabditis elegans, development is spatially and temporally determined; adult cell number and anatomy are nearly invariant. Cohorts of isogenic animals synchronously express genes that cause individuals to molt to successive larval stages within a few hours of one other. Yet, even isogenic cohorts cultured in homogenous, liquid environments show 20-fold variation in individual lifespan. Five years ago, we (Johnson lab) found (REA et al. 2005) in isogenic populations that level of expression of a heat-shock promoter-GFP fusion construct (Phsp-16.2::gfp) after mild heat stress predicts subsequent lifespan. Thus, Phsp-16.2 reporter expression on the first day of adult life is a physiological variable (or 'biomarker') predictive of subsequent lifespan. In metazoans, the components and mechanism(s) underlying expression variation of the Phsp-16.2::gfp biomarker (or any other gene) remain mostly unknown. For that reason we have initiated work on Phsp-16.2::gfp expression modeled on previous work in S.cerevisiae. In these yeast studies, we (Brent lab) and others have dissected and quantified different contributions to 'stochastic' variation in gene expression and on cell fate decisions (COLMAN-LERNER et al. 2005). As in these studies, we are quantifying gene expression and variation, dissecting the different contributions to those quantities, and using genetics to understand control of the individual sources of variation. To do so, we measure expression of different-colored fluorescent reporter proteins, under the control of identical or of different promoters, quantify components of the variation based on simple mathematical models, and use the results to guide subsequent hypothesis-directed experiments. At this point, we have some genetic data identifying physiological subsystems that control the amount of worm-to-worm variation in gene expression. We have used defined site, single copy reporter integration technology (MosSCI technology) to determine how variance in gene expression manifests when using single copy reporter genes vs tandem arrays and to minimize variation in gene expression due to chromosomal position and silencing. We are using these reporters to quantify the amount of worm-to-worm variation due to intrinsic noise and that due to the different physiological subsystems. Colman-Lerner,A., A. Gordon,E. Serra, T. Chin, O.Resnekov et al., 2005 Regulated cell-to-cellvariation in a cell-fate decision system. Nature 437: 699-706. Rea, S.L., D. Wu,J. R. Cypser, J. W.Vaupel and T. E.Johnson, 2005 Astress-sensitive reporter predicts longevity in isogenic populations of Caenorhabditis elegans. Nat Genet 37: 894-898.
[
International Worm Meeting,
2019]
The maintenance of males at intermediate frequencies is an important evolutionary problem. Several species of Caenorhabditis nematodes have evolved the androdioecious mating system, where selfing hermaphrodites and males coexist. They reproduce mostly through self-fertilization, but can also outcross. While selfing produces XX hermaphrodites, cross-fertilization produces 50% XO male progeny. Thus, male mating success dictates the sex ratio [1]. Here, we focus on the contribution of the male secreted short (mss) gene family to male mating success, sex ratio, and population growth. The mss family is essential for full sperm competitiveness in gonochoristic species, but has been lost in parallel in androdioecious species [2]. Using a transgene to restore mss function to the androdioecious C. briggsae, we examined how mating system and population subdivision influence the fitness of the mss+ genotype. Consistent with theoretical expectations, mss+ is sufficient to increase male frequency and depress population growth in genetically homogenous androdioecious populations. When mss+ and mss-null (i.e. wild-type) genotypes compete, mss+ is positively selected in both mixed-mating and strictly outcrossing situations, though more strongly in the latter. Thus, while sexual mode alone affects the fitness of mss+, it is insufficient to explain its parallel loss. We propose that the lack of inbreeding depression [3] and the strong subdivision that characterize natural Caenorhabditis populations [4] impose selection on sex ratio that makes loss of mss adaptive. By reducing, but not completely eliminating outcrossing, loss of the mss genes tunes the sex ratio to its new optimum after self-fertility is established. 1. Stewart AD, Phillips PC (2002) Genetics 160: 975 2. Yin et al. (2018) Science 359: 55 3. Dolgin et al. (2007) Evolution 61: 1339 4. Kiontke KC, et al. (2011) BMC Evol Biol 11: 339
[
International Worm Meeting,
2019]
Extracellular vesicles (EVs) are membrane wrapped structures containing proteins, RNAs, lipids, and metabolites that are released from most if not all cell types to mediate intercellular communication in physiological and pathological conditions. EVs fall into subclasses based on their mode of biogenesis. Exosomes are released following fusion of multivesicular bodies (MVBs) with the plasma membrane, while microvesicles form directly from plasma membrane budding. Our goal is to use C. elegans to identify proteins required for EV biogenesis in vivo. EVs are released from the cilia of the male specific cephalic male (CEM) neurons in the head and hook B type (HOB) and bilateral ray B type neurons (RnB) in the tail. These EVs are likely microvesicles as there is no evidence of MVBs in these neurons1. We discovered that the ion channel CLHM-1 is cargo in EVs released from C. elegans ciliated male sensory neurons by performing high resolution imaging of animals expressing functional GFP-tagged CLHM-1 at endogenous levels. Remarkably, when we co-expressed tdTomato-tagged CLHM-1 with GFP-tagged PKD-2, a known EV cargo protein expressed in the same neurons1, we rarely observed colocalization of the fluorescent proteins in vesicles, suggesting that CLHM-1 and PKD-2 are in discrete EV subpopulations. We are using the power of genetics to manipulate candidate EV biogenesis pathways to identify factors required for release of CLHM-1 containing EVs. Lipid asymmetry between the inner and outer leaflets of the plasma membrane induces curvature which could drive microvesicle release. Type IV-ATPase flippases translocate phospholipids from the outer to the inner leaflet to maintain bilayer asymmetry, while scramblases disrupt membrane asymmetry. We are determining if the flippases TAT-1, TAT-3, and TAT-6 as well as the scramblases ANOH-1 and SCRM-4 play a role in the biogenesis of one or both fluorescently marked EV subpopulations derived from male ciliated neurons. 1. Wang J, Silva M, Haas LA, Morsci NS, Nguyen KC, Hall DH, Barr MM. (2014) C. elegans ciliated sensory neurons release extracellular vesicles that function in animal communication. Curr Biol. 24(5):519-25.