Aging of the Reproductive System and of Germline Stem Cells in Caenorhabditis

C. elegans hermaphrodites display dramatic age-related decline of reproduction early in life while somatic functions are still robust. To understand why the germline fails so early, we analyzed the assembly line of oocyte production that generates fertilized eggs in mated hermaphrodites with sufficient sperm. Aging germlines displayed both sporadic and population-wide changes. A small fraction of aging animals displayed endomitotic oocytes in the germline and other defects. By contrast, all animals displayed age-related decreases in germline size and function. As early as day 3 of adulthood, animals displayed fewer stem cells and a slower cell cycle, which combine to substantially decrease progenitor zone output. The C. elegans germline is the only adult tissue that contains stem cells, allowing the analysis of stem cells in aging. To investigate the mechanism of the decrease in stem cell number, we analyzed the Notch signaling pathway. The Notch effectors LST-1 and SYGL-1 displayed age-related decreases in expression domains, suggesting a role for Notch signaling in germline aging. The results indicate that while sporadic defects account for the sterility of some animals, population-wide changes account for the overall pattern of reproductive aging. Moderate caloric restriction and specific mutations in the insulin pathway result in extended reproductive span, albeit with decreased peak reproduction. We found that while the germline of these mutant animals started out smaller and meiotic entry was lower than in wild-type, the slope of decline was more gradual than in wild-type. This was consistent with the hypothesis that an age-related decrease in progenitor function leads to a decrease in meiotic entry and causes the age-related decrease in progeny production. We also identified mid-life reproductive improvement, a pattern of increased progeny production on days 5-7 of adulthood in che-3(lf) and sygl-1(tg), and found that certain mutants extended lifespan without compromising early progeny production and that other mutants increased reproductive ability without extending lifespan. These results showed that while reproductive function and somatic lifespan are inter-linked, they are also separable

Strategies for delivery of CRISPR/Cas-mediated genome editing to obtain edited plants directly without transgene integration

Increased understanding of plant genetics and the development of powerful and easier-to-use gene editing tools over the past century have revolutionized humankind’s ability to deliver precise genotypes in crops. Plant transformation techniques are well developed for making transgenic varieties in certain crops and model organisms, yet reagent delivery and plant regeneration remain key bottlenecks to applying the technology of gene editing to most crops. Typical plant transformation protocols to produce transgenic, genetically modified (GM) varieties rely on transgenes, chemical selection, and tissue culture. Typical protocols to make gene edited (GE) varieties also use transgenes, even though these may be undesirable in the final crop product. In some crops, the transgenes are routinely segregated away during meiosis by performing crosses, and thus only a minor concern. In other crops, particularly those propagated vegetatively, complex hybrids, or crops with long generation times, such crosses are impractical or impossible. This review highlights diverse strategies to deliver CRISPR/Cas gene editing reagents to regenerable plant cells and to recover edited plants without unwanted integration of transgenes. Some examples include delivering DNA-free gene editing reagents such as ribonucleoproteins or mRNA, relying on reagent expression from non-integrated DNA, using novel delivery mechanisms such as viruses or nanoparticles, using unconventional selection methods to avoid integration of transgenes, and/or avoiding tissue culture altogether. These methods are advancing rapidly and already enabling crop scientists to make use of the precision of CRISPR gene editing tools

Reproductive aging in Caenorhabditis elegans: From molecules to ecology

Aging animals display a broad range of progressive degenerative changes, and one of the most fascinating is the decline of female reproductive function. In the model organis

Genetic analysis of reproductive aging in <i>C. elegans</i>.

<p>(A) <i>C. elegans</i> displays a series of age-related degenerative changes including loss of self-fertile reproduction (red), mated reproduction (blue), pharyngeal pumping (green), and survival (black). (B) Three major discovery approaches have been used to identify genes that influence reproductive aging.</p

A forward genetic screen for mutations that affect zinc-activated transcription of the <i>cdf-2</i> promoter.

<p><b>(A)</b> A diagram (not to scale) of the <i>cdf-2p</i>::<i>gfp</i> transcriptional reporter construct containing the <i>cdf-2</i> promoter (black line, 1,371bp upstream of the ATG start codon) fused to the coding region of green fluorescence protein (green box). The <i>cdf-2</i> promoter contains a high zinc activation (HZA) enhancer element (orange box, 194 bp upstream of the ATG start codon) [<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2000094#pbio.2000094.ref015" target="_blank">15</a>]. This construct was integrated into the genome of <i>C</i>. <i>elegans</i> to generate transgenic strain WU1391 that functions as a high zinc reporter strain. <b>(B)</b> WU1391 transgenic animals containing the <i>cdf-2p</i>::<i>gfp</i> transcriptional reporter were cultured with standard Noble agar minimal medium (NAMM) (0 μM Supplemental Zinc) or NAMM supplemented with 200 μM supplemental zinc. GFP fluorescence was observed with a compound microscope, and representative images are shown. Animals were oriented with the long axis horizontally—the intestine is a prominent tubular structure spanning the length of the animal, and it is outlined with a dotted white line. Animals cultured in medium with no supplemental zinc displayed low-level fluorescence that is a combination of autofluorescence due to gut granules and low level expression of the <i>cdf-2</i> promoter. By contrast, intestinal fluorescence is prominent in animals cultured in zinc-supplemented medium. Scale bars are approximately 100 μm. <b>(C)</b> A flow chart of the forward genetic screen. WU1391 transgenic hermaphrodites (P₀) were mutagenized by ethyl methanesulfonate (EMS) and allowed to self-fertilize for two generations. F₂ self progeny that are homozygous for newly induced mutations were analyzed. <u><i>Z</i></u>inc-<u><i>a</i></u>ctivated <u><i>t</i></u>ranscription-<u><i>c</i></u>onstitutive (Zat-c) mutants were isolated by screening for animals that displayed fluorescence when cultured on medium with no supplemental zinc. <u><i>Z</i></u>inc-<u><i>a</i></u>ctivated <u><i>t</i></u>ranscription-<u><i>d</i></u>eficient (Zat-d) mutants were isolated by screening for animals that did not display fluorescence when cultured on medium with 200 μM supplemental zinc. <b>(D)</b> WU1391, the Zat-d mutant strains (<i>am279</i>, <i>am280</i>, <i>am286</i>, <i>am287</i>, and <i>am288</i>) and the Zat-c mutant strain (<i>am285</i>) were cultured on medium with 0 or 200 μM supplemental zinc. Fluorescence intensity was quantified using microscopy and is expressed in arbitrary units (A.U.). Bars represent the average fluorescence intensity +/- standard deviation (S.D.) (<i>n</i> = 12–18 animals). Compared to the unmutagenized WU1391 starting strain, all five Zat-d mutant strains displayed significantly reduced fluorescence intensity when cultured with 200 μM supplemental zinc; the one Zat-c mutant strain displayed significantly increased fluorescence intensity when cultured with 0 μM supplemental zinc (*, <i>p</i> < 0.05). The Zat-c mutant strain cultured on 200 μM supplemental zinc displayed significantly increased levels of GFP fluorescence compared to the Zat-c mutant strain cultured on 0 μM supplemental zinc and WU1391 animals cultured on 200 μM supplemental zinc. Thus, the Zat-c mutant strain retains zinc-activated transcription while displaying higher baseline expression levels compared to wild type. <b>(E)</b> Upper, physical map of a portion of <i>C</i>. <i>elegans</i> linkage group X (LGX) with loci positions in base pairs (k = thousand). Zat-d and Zat-c mutations were positioned between <i>egl-15</i> and <i>sma-5</i>. Lower, <i>hizr-1</i> locus (not to scale). Open boxes are exons, shaded regions are untranslated, orange box is the HZA enhancer, black bars locate the DNA-binding (DBD) and ligand-binding (LBD) domains, and lines locate mutations.</p

The Nuclear Receptor HIZR-1 Uses Zinc as a Ligand to Mediate Homeostasis in Response to High Zinc

<div><p>Nuclear receptors were originally defined as endocrine sensors in humans, leading to the identification of the nuclear receptor superfamily. Despite intensive efforts, most nuclear receptors have no known ligand, suggesting new ligand classes remain to be discovered. Furthermore, nuclear receptors are encoded in the genomes of primitive organisms that lack endocrine signaling, suggesting the primordial function may have been environmental sensing. Here we describe a novel <i>Caenorhabditis elegans</i> nuclear receptor, HIZR-1, that is a high zinc sensor in an animal and the master regulator of high zinc homeostasis. The essential micronutrient zinc acts as a HIZR-1 ligand, and activated HIZR-1 increases transcription of genes that promote zinc efflux and storage. The results identify zinc as the first inorganic molecule to function as a physiological ligand for a nuclear receptor and direct environmental sensing as a novel function of nuclear receptors.</p></div

A Laboratory And Simulation Platform To Integrate Individual Life History Traits And Population Dynamics

Understanding populations is important as they are a fundamental level of biological organization. Individual traits such as aging and lifespan interact in complex ways to determine birth and death, and thereby influence population dynamics; however, we lack a deep understanding of the relationships between individual traits and population dynamics. To address this challenge, we established a laboratory population using the model organism Caenorhabditis elegans and an individual-based computational simulation informed by measurements of real worms. The simulation realistically models individual worms and the behavior of the laboratory population. To elucidate the role of aging in population dynamics, we analyzed old age as a cause of death and showed, using computer simulations, that it was influenced by maximum lifespan, rate of adult culling and progeny number/food stability. Notably, populations displayed a tipping point for aging as the primary cause of adult death. Our work establishes a conceptual framework that could be used for better understanding why certain animals die of old age in the wild

HIZR-1 regulates high zinc homeostasis in the <i>C</i>. <i>elegans</i> intestine.

<p><b>(A)</b> Genetic model. High levels of zinc promote HIZR-1 activity and transcriptional activation of multiple genes including <i>cdf-2</i>, <i>ttm-1b</i> and <i>hizr-1</i>. Increased levels of <i>cdf-2</i> and <i>ttm-1b</i> mRNA promote increased levels of CDF-2 and TTM-1B protein, which reduce levels of cytoplasmic zinc in a parallel negative feedback circuit. Increased levels of <i>hizr-1</i> mRNA promotes increased levels of HIZR-1 protein, creating a positive feedback circuit that enhances the negative feedback system. <b>(B)</b> Molecular model. Dietary zinc (Z) enters intestinal cells, binds the LBD of HIZR-1 and promotes nuclear accumulation, HZA enhancer binding, and transcriptional activation. The nuclear accumulation of HIZR-1 could result from increased HIZR-1 protein levels due to autoregulation and/or translocation of HIZR-1 from the cytoplasm to the nucleus. Increased levels of CDF-2 and TTM-1B promote zinc detoxification by sequestration in lysosome-related organelles [<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2000094#pbio.2000094.ref013" target="_blank">13</a>] and excretion into the intestinal lumen [<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2000094#pbio.2000094.ref014" target="_blank">14</a>], respectively. Increased levels of HIZR-1 promote homeostasis by a positive feedback circuit.</p