Study of extracellular matrix synthesis in C. elegans

The epithelial monolayer of cells surrounding the animal, the hypodermis, Synthesises five cuticles during the nematode life cycle. The first cuticle is formed within the egg, prior to hatching, and the remainder towards the end of each larval stage. Because of the structural role of the cuticle, mutations in genes involved in assembly of this ECM can cause a spectrum of effects from lethality late in embryogenesis to alterations in the nematode shape. The severity of phenotype correlates with the severity of cuticle synthesis defects. Accordingly, two distinct mutant alleles that cause death after embryonic elongation, possibly due to failure in synthesising an intact cuticle, were characterised . One mutant, ij15, was isolated from a forward genetic screen previously performed (I. Johnstone, Glasgow University, Glasgow, UK). ij15 defines mutationally the gene stc-1, which encodes a HSP70-like protein possibly localised in the secretory pathway. The other mutant, h402, defines mutationally the gene let-607. A second let-607 allele, h189, which results in larval lethaity at the L2 stage was also analysed in this study. let-607 corresponds to the predicted gene F57B10.1, which encodes a putative bZIP transcription factor. Both stc-1 and let-607 are expressed in the hypodermis at all developmental stages. Furthermore, disruption of the function of either stc-1 or let-607 by mutation or RNAi affects cuticle synthesis in different ways. Thus, stc-1 and let-607 encode for a HSP70-like protein and a putative bZIP transcription factor required for synthesis of the cuticular ECM in C. elegans. In addition, this study defines C. elegans mutant phenotypes that can be used as indicators for gene products with controlling roles in the synthesis of this ECM

MicroRNAs in C. elegans Aging: Molecular Insurance for Robustness?

The last decade has witnessed a revolution in our appreciation of the extensive regulatory gene expression networks modulated by small untranslated RNAs. microRNAs (miRNAs), ~22 nt RNAs that bind imperfectly to partially homologous sites on target mRNAs to regulate transcript expression, are now known to influence a broad range of biological processes germane to development, homeostatic regulation and disease. It has been proposed that miRNAs ensure biological robustness, and aging has been described as a progressive loss of system and cellular robustness, but relatively little work to date has addressed roles of miRNAs in longevity and healthspan (the period of youthful vigor and disease resistance that precedes debilitating decline in basic functions). The C. elegans model is highly suitable for testing hypotheses regarding miRNA impact on aging biology: the lifespan of the animal is approximately three weeks, there exist a wealth of genetic mutations that alter lifespan through characterized pathways, biomarkers that report strong healthspan have been defined, and many miRNA genes have been identified, expression-profiled, and knocked out. 50/114 C. elegans miRNAs change in abundance during adult life, suggesting significant potential to modulate healthspan and lifespan. Indeed, miRNA lin-4 has been elegantly shown to influence lifespan and healthspan via its lin-14 mRNA target and the insulin signaling pathway. 27 of the C. elegans age-regulated miRNAs have sequence similarity with both fly and human miRNAs. We review current understanding of a field poised to reveal major insights into potentially conserved miRNA-regulated networks that modulate aging

The biogenesis and characterization of mammalian microRNAs of mirtron origin

Mirtrons, short hairpin pre-microRNA (miRNA) mimics directly produced by intronic splicing, have recently been identified and experimentally confirmed in invertebrates. While there is evidence to suggest several mammalian miRNAs have mirtron origins, this has yet to be experimentally demonstrated. Here, we characterize the biogenesis of mammalian mirtrons by ectopic expression of splicing-dependent mirtron precursors. The putative mirtrons hsa-miR-877, hsa-miR-1226 and mmu-miR-1224 were designed as introns within eGFP. Correct splicing and function of these sequences as introns was shown through eGFP fluorescence and RT–PCR, while all mirtrons suppressed perfectly complementary luciferase reporter targets to levels similar to that of corresponding independently expressed pre-miRNA controls. Splicing-deficient mutants and disruption of key steps in miRNA biogenesis demonstrated that mirtron-mediated gene knockdown was splicing-dependent, Drosha-independent and had variable dependence on RNAi pathway elements following pre-miRNA formation. The silencing effect of hsa-miR-877 was further demonstrated to be mediated by the generation of short anti-sense RNA species expressed with low abundance. Finally, the mammalian mirtron hsa-miR-877 was shown to reduce mRNA levels of an endogenous transcript containing hsa-miR-877 target sites in neuronal SH-SY5Y cells. This work confirms the mirtron origins of three mammalian miRNAs and suggests that they are a functional class of splicing-dependent miRNAs which are physiologically active

MicroRNA Predictors of Longevity in Caenorhabditis elegans

Neither genetic nor environmental factors fully account for variability in individual longevity: genetically identical invertebrates in homogenous environments often experience no less variability in lifespan than outbred human populations. Such variability is often assumed to result from stochasticity in damage accumulation over time; however, the identification of early-life gene expression states that predict future longevity would suggest that lifespan is least in part epigenetically determined. Such “biomarkers of aging,” genetic or otherwise, nevertheless remain rare. In this work, we sought early-life differences in organismal robustness in unperturbed individuals and examined the utility of microRNAs, known regulators of lifespan, development, and robustness, as aging biomarkers. We quantitatively examined Caenorhabditis elegans reared individually in a novel apparatus and observed throughout their lives. Early-to-mid–adulthood measures of homeostatic ability jointly predict 62% of longevity variability. Though correlated, markers of growth/muscle maintenance and of metabolic by-products (“age pigments”) report independently on lifespan, suggesting that graceful aging is not a single process. We further identified three microRNAs in which early-adulthood expression patterns individually predict up to 47% of lifespan differences. Though expression of each increases throughout this time, mir-71 and mir-246 correlate with lifespan, while mir-239 anti-correlates. Two of these three microRNA “biomarkers of aging” act upstream in insulin/IGF-1–like signaling (IIS) and other known longevity pathways, thus we infer that these microRNAs not only report on but also likely determine longevity. Thus, fluctuations in early-life IIS, due to variation in these microRNAs and from other causes, may determine individual lifespan

Sequence Relationships among C. elegans, D. melanogaster and Human microRNAs Highlight the Extensive Conservation of microRNAs in Biology

microRNAs act in a prevalent and conserved post-transcriptional gene regulatory mechanism that impacts development, homeostasis and disease, yet biological functions for the vast majority of miRNAs remain unknown. Given the power of invertebrate genetics to promote rapid evaluation of miRNA function, recently expanded miRNA identifications (miRBase 10.1), and the importance of assessing potential functional redundancies within and between species, we evaluated miRNA sequence relationships by 5′ end match and overall homology criteria to compile a snapshot overview of miRNA families within the C. elegans and D. melanogaster genomes that includes their identified human counterparts. This compilation expands literature documentation of both the number of families and the number of family members, within and between nematode and fly models, and highlights sequences conserved between species pairs or among nematodes, flies and humans. Themes that emerge include the substantial potential for functional redundancy of miRNA sequences within species (84/139 C. elegans miRNAs and 70/152 D. melanogaster miRNAs share significant homology with other miRNAs encoded by their respective genomes), and the striking extent to which miRNAs are conserved across species—over half (73/139) C. elegans miRNAs share sequence homology with miRNAs encoded also in both fly and human genomes. This summary analysis of mature miRNA sequence relationships provides a quickly accessible resource that should facilitate functional and evolutionary analyses of miRNAs and miRNA families

CeleST: computer vision software for quantitative analysis of C. elegans swim behavior reveals novel features of locomotion.

In the effort to define genes and specific neuronal circuits that control behavior and plasticity, the capacity for high-precision automated analysis of behavior is essential. We report on comprehensive computer vision software for analysis of swimming locomotion of C. elegans, a simple animal model initially developed to facilitate elaboration of genetic influences on behavior. C. elegans swim test software CeleST tracks swimming of multiple animals, measures 10 novel parameters of swim behavior that can fully report dynamic changes in posture and speed, and generates data in several analysis formats, complete with statistics. Our measures of swim locomotion utilize a deformable model approach and a novel mathematical analysis of curvature maps that enable even irregular patterns and dynamic changes to be scored without need for thresholding or dropping outlier swimmers from study. Operation of CeleST is mostly automated and only requires minimal investigator interventions, such as the selection of videotaped swim trials and choice of data output format. Data can be analyzed from the level of the single animal to populations of thousands. We document how the CeleST program reveals unexpected preferences for specific swim "gaits" in wild-type C. elegans, uncovers previously unknown mutant phenotypes, efficiently tracks changes in aging populations, and distinguishes "graceful" from poor aging. The sensitivity, dynamic range, and comprehensive nature of CeleST measures elevate swim locomotion analysis to a new level of ease, economy, and detail that enables behavioral plasticity resulting from genetic, cellular, or experience manipulation to be analyzed in ways not previously possible

CeleST reveals novel information on aging phenotypes.

<p><b>A–J</b>, Age-associated changes in swimming parameters in wild-type adults. <b>A</b>, Wave initiation rate; <b>B</b>, Body wave number; <b>C</b>, Asymmetry; <b>D</b>, Stretch; <b>E</b>, Attenuation; <b>F</b>, Reverse swimming; <b>G</b>, Curling; <b>H</b>, Travel speed; <b>I</b>, Brush stroke; and <b>J</b>, Activity index. from 9 independent trials, for each age day 4 and day 11. Data for ages ranging from day 4 to day 20 are presented in <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1003702#pcbi.1003702.s004" target="_blank">Figure S4</a>. <b>K–M</b>, CeleST reports great differences in graceful agers vs. poor agers for measures that change with age. We selected animals that appeared to have robust crawling capacity (Class A, graceful agers) and those that had decrepit crawling capacity (Class C, poor agers) at day 11 and compared swim behavior. <b>K</b>, Activity index; <b>L</b>, Asymmetry; <b>M</b>, Attenuation. from 3 independent trials, for each class. Data for all ten measures in this series are shown in <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1003702#pcbi.1003702.s005" target="_blank">Figure S5</a>. <b>N</b>, Locomotory changes under life-extending and progeric insulin signaling pathway manipulation suggest complex influences of signaling over the lifetime. Activity index, WT: blue line (middle dashed line); long lived <i>age-1(hx546)</i>: green (top line); short-lived <i>daf-16(mgDf50)</i>: red (bottom line). Note that the <i>age-1</i> mutant has a higher activity index in young adult life as compared to WT, which suggests differences in swim performance are not limited to aging. Also, at day 15, WT and <i>age-1</i> scores appear increased, which we suggest reflects the preferential death of the poorest swimmers, rather than an actual increase in average swimming of individuals. in each data point from 4 independent trials. Data on all measures are presented in <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1003702#pcbi.1003702.s006" target="_blank">Figure S6</a>. Error bars show SEM, *** , **** .</p