Ow, Maria C. et al. (2020) MicroPubl Biol

Ow, Maria C., Hall, Sarah E.

[MicroPubl Biol, 2020]

During its first larval stage (L1), the nematode C. elegans makes a critical decision regarding its developmental trajectory based on environmental conditions. Poor conditions such food scarcity, crowding, or high temperatures promote the entry into a stress-resistant, non-aging, diapause stage named dauer where they can survive for months. Upon improved conditions, dauer larvae resume development as postdauer (PD) L4 larvae and continue to reproductive adulthood as PD adults (Cassada and Russell, 1975). If conditions are favorable, L1 larvae proceed through additional larval stages (L2-L4) until reaching reproductive adulthood (control adults or CON) (Sulston and Horvitz, 1977). We have previously shown that PD adults retain a cellular memory of their early life experience that results in stress-specific transcriptome changes that promote alterations in their life history traits (Hall et al. 2010, 2013; Ow et al. 2018). For instance, wild-type CON adults and PD adults that experienced early-life starvation exhibit reproductive differences that are manifested as decreased brood size in PD animals relative to CON adults. Reproduction in self-fertilizing hermaphrodites is sperm-limited; thus, brood size differences can result from varying numbers of sperm produced by individual animals. We showed previously that postdauers animals that experienced early-life starvation exhibit a significant delay in germline proliferation during the period when sperm are produced compared to control animals of comparable developmental stage. This observation suggests that the decrease in postdauers adult brood size is consistent with a reduction in sperm number, which we have referred to as reproductive plasticity (Ow et al. 2018). The standard wild-type N2 strain was first cultivated in the laboratory over five decades ago, resulting in the fixation of random mutations over thousands of generations in laboratory conditions atypical to what are experienced by natural populations (Sterken et al. 2015). We wondered whether the reproductive plasticity observed in the canonical laboratory wild-type N2 strain is an adaptive trait acquired over time as a result of laboratory conditions, or represents a conserved mechanistic response to starvation stress. We assessed CON and PD brood sizes of six natural isolates representing various branches of the C. elegans phylogenetic tree (Andersen et al. 2012). CON L4 larvae were obtained from a continuously growing mixed population of worms cultured on NGM plates at 20oC. To obtain PD L4 larvae, well-fed worms were first starved on NGM plates at 20oC for about one week until dauers were visible, then worms were collected and incubated for 30 minutes in 1% SDS (sodium dodecyl sulfate) at room temperature with gentle rotation. Because dauers have suspended pharyngeal pumping (Cassada and Russell, 1975), they survive the 1% SDS treatment that would otherwise be lethal if the detergent were ingested. Dauers were then washed with M9 buffer to rinse away the SDS and placed onto seeded NGM plates at 20oC to promote dauer exit. Once the dauers had developed into postdauer L4s, brood size assays were performed in parallel with their CON counterparts by randomly singling out L4 larvae onto seeded 35 mm NGM plates at 20oC, transferring daily them to fresh plates until egg laying ceased, and counting the number of surviving progeny. These surviving progenies were counted as young adults (four days after the mothers were transferred to fresh NGM plates). We found that certain wild isolates, (e.g. AF16 in Figure 1), had the proclivity to crawl to the side of an NGM plate assay plate and desiccate. We thus censored the data for any animal that died before the end of the egg-laying period. We found that four out of six natural isolates (AB1, CB4856, ED3040, and TR403) displayed a significant reduction in brood size in PD adults relative to CON adults similar to N2. The strains, JU440 and KR314, did not exhibit reproductive plasticity and had a similar number of progeny between CON and PD adults (Figure 1). Interestingly, when we measured the brood size in CON and PD adults of another nematode species closely related to C. elegans, C. briggsae AF16, it displayed a modest but statistically insignificant decrease in PD brood size compared to CON adults (Figure 1). Is the reproductive plasticity between CON and PD a reflection of different mating strategies among Caenorhabditis strains? The spontaneous male frequency in the N2 strain is a low ~0.1%, and reproduction is usually achieved through hermaphrodite self-fertilization (Chasnov and Chow, 2002). However, we found that one natural isolate, CB4856, which harbors a higher frequency of males in their population than the laboratory N2 strain (Wegewitz et al. 2008), also exhibits reproductive plasticity between CON and PD. This observation suggests that reproductive plasticity between CON and PD adults may not be simply due to changes in male frequency and levels of mating. The distinction between strains exhibiting adult reproductive plasticity and those that do not is not immediately obvious when comparing phylogenetic proximity or strain isolation date (Andersen et al. 2012). Thus, additional experiments would be necessary to determine the genetic loci in N2 Bristol and the wild isolates that modulate this reproductive trait. Taken together, these results suggest that reproductive plasticity observed in C. elegans is a naturally occurring developmental trait rather than an adaptive trait stemming from decades of cultivation in a laboratory.

Ow, Maria C. et al. (2013) International Worm Meeting "Genome-wide study of stress-responsive microRNA and mRNA transcriptomes in C. elegans."

Ambros, Victor, Veksler-Lublinsky, Isana, Ow, Maria C.

[International Worm Meeting, 2013]

All organisms must cope with stress and have evolved various means to respond to detrimental changes in their environment. Because most of the pathways involved in stress response are conserved throughout evolution, studying stress response in C. elegans is likely to result in crucial insights in understanding the pathology and treatment of human disorders. MicroRNAs (miRNAs) are an abundant class of small non-coding RNAs that regulate gene expression by imperfectly base-pairing to the 3'UTR of their target mRNAs. MiRNAs are intimately involved in broad aspects of animal development. Their misregulation can result in the onset of a number of pathophysiological conditions, including heart disease, diabetes, and cancer. Given the prominent role of miRNAs in nearly all aspects of biology, we predict that they also play a role in stress response. In this study we employ genome-wide methods, including small RNA deep sequencing and mRNA transcript microarrays, to study the response of C. elegans to various stresses including oxidative stress, heat stress, and exposure to bacterial pathogens. We employ bioinformatics to determine stress-responsive miRNAs and mRNAs and identify stress-mediated gene regulatory patterns. Our analyses suggest the existence of regulatory patterns common to all stresses as well as stress-specific sub-systems.

Maria C Ow et al. (2005) International Worm Meeting "Regulation of lin-4 expression in C. elegans."

Victor R Ambros, Maria C Ow, Phillip Olsen, Scott Silverman

[International Worm Meeting, 2005]

The lin-4 gene in C. elegans encodes the founding member of a class of small noncoding RNAs known as microRNAs that post-transcriptionally regulate gene expression by antisense base pairing. We have conducted a yeast-one-hybrid screen to identify regulators of lin-4 expression and have identified Y11D7A.12, a novel protein with a Zinc-finger FLYWHC DNA binding domain, as an interactor of the DNA region upstream of lin-4. To ascertain the effect of RNAi-by-feeding of Y11D7A.12 on the transcription of lin-4, we generated transgenic animals encoding 2.4 kb of the lin-4 upstream region fused to a gfp reporter. These transgenic animals recapitulate the native expression of lin-4; gfp expression is upregulated during the mid-L1 larval stage after the larvae have commenced feeding. RNAi of Y11D7A.12 resulted in no discernable phenotype. However, simultaneous RNAi of both Y11D7A.12 and its C. elegans paralog, C26E6.2, in RNAi-hypersensitive animals resulted in a low penetrance of pleiotropic phenotypes that include premature transcription of lin-4 during embryogenesis, morphological abnormal larvae, and larval lethality. This suggests that Y11D7A.12, together with C26E6.2, is involved in various embryonic and postembryonic processes and may act as a negative temporal regulator of lin-4 transcription.

Maria C. Ow et al. (2007) International Worm Meeting "Transcription factors controlling the timing of microRNA gene expression in C. elegans."

Natalia J. Martinez, Maria C. Ow, A. J. Walhout, Phillip Olsen, Scott Silverman, BARBARA CONRADT, Victor R. Ambros

[International Worm Meeting, 2007]

MicroRNAs are an abundant class of small non-coding RNAs that regulate gene expression post-transcriptionally via anti-sense base pairing. Although microRNAs function in regulating a number of cellular events, very little is known about how the expression of microRNAs is regulated. We have conducted a yeast-one-hybrid screen to identify transcription factors that bind to the upstream region of the founding member of the microRNAs, lin-4, and have identified a novel protein with a Zinc-finger FLYWCH DNA binding domain as an interactor of the DNA region upstream of lin-4. RNAi-by-feeding of Y11D7A.12 results in the premature expression of lin-4 during the embryonic stage as assessed by a lin-4 transcriptional GFP reporter and Northern blot analysis. The simultaneous RNAi of both Y11D7A.12 and its C. elegans paralog, C26E6.2, enhances this precocious lin-4 expression phenotype. A deletion mutant of Y11D7A.12 also results in the premature expression of lin-4 as well as a low penetrance of morphological abnormal L1 larvae and early larval lethality. The double deletion mutant of Y11D7A.12 and C26E6.2 results in the complete penetrance of early larval lethality. This lethal phenotype is not suppressed by a lin-4(lf) mutation, suggesting that Y11D7A.12 and C26E6.2 regulate the expression of other genes in addition to lin-4. A more comprehensive yeast-one-hybrid screen using Y11D7A.12 and C26E6.2 suggest that these proteins also have roles in the regulation of additional microRNAs. GFP reporter transgenes and developmental Westerns indicate that Y11D7A1.2 and C26E6.2 are expressed primarily in embryos, suggesting that they function as negative temporal regulators of their target microRNAs early in development.

Sims, Jennie et al. (2015) International Worm Meeting "Developmental history regulates olfactory behavior via RNAi pathways."

Ow, Maria C., Nishiguchi, Mailyn, Hall, Sarah E., Sims, Jennie, Sengupta, Piali

[International Worm Meeting, 2015]

Environmental stress early in development can impact adult phenotypes through programmed changes in gene expression. C. elegans larvae respond to environmental stress by entering the alternative dauer diapause pathway, and resume development once conditions improve (postdauers). Our previous work showed that postdauer adults retain a cellular memory of their developmental history through changes in gene expression, genome-wide chromatin states, small RNA profiles, and life history traits when compared to animals that bypassed the dauer stage (controls). However, little is known about the mechanisms that establish and maintain these changes in response to developmental history. We have identified the osm-9 TRPV channel gene as a target of environmental programming. OSM-9 is expressed in multiple sensory neurons and is essential for mechanosensory, osmosensory, and chemosensory behaviors. In control adults, gfp driven by ~350 bp of osm-9 upstream regulatory sequences was expressed in the ADL and AWA neurons, whereas gfp was only expressed in AWA neurons of postdauer adults. Analysis of the osm-9 upstream regulatory sequence yielded a cis-acting motif that is necessary for the downregulation of osm-9 in ADL, and is competitively bound by the negative regulator, DAF-3 SMAD, and unknown activators. In addition, we found that the ADL-mediated and osm-9 dependent ascr#3 avoidance behavior is significantly reduced in wildtype postdauer adults compared to controls, indicating that the endogenous osm-9 gene is downregulated similarly to the gfp reporter. Using the ascr#3 avoidance assay, we investigated the mechanism of osm-9 regulation by testing the behavior of control and postdauer animals in strains carrying mutations in dauer formation, RNAi, and chromatin remodeling genes. Our results indicated that the differential expression of osm-9 was mediated by TGF-beta signaling and required functional chromatin remodeling pathways. Moreover, we found that the Mutator and ERGO-1 26G RNAi pathways are also required for the regulation of osm-9 expression. We showed that MUT-16 modulates the expression of daf-3, and is required in ADL neurons for the differential expression of osm-9. Together, our results suggest that RNAi pathways regulate osm-9 expression indirectly via modulation of TGF-beta signaling, and that the altered expression pattern is maintained by the chromatin state at the osm-9 locus. Our results describe an elegant mechanism by which developmental experience influences adult phenotypes by establishing and maintaining transcriptional changes via RNAi and chromatin remodeling pathways.

Nichitean, Alexandra M. et al. (2021) International Worm Meeting "Oleic acid modulates reproductive plasticity via DAF-12 in postdauer adults"

Hall, Sarah E., Ow, Maria C., Compere, Frances V., Nichitean, Alexandra M.

[International Worm Meeting, 2021]

Environmental stress experienced during critical periods of development has been shown to result in reproductive plasticity in both plants and animals, although the molecular mechanisms regulating these phenotypes are not well understood. In previous work, we showed that different environmental stresses experienced during early development in Caenorhabditis elegans resulted in distinct reproductive outcomes. C. elegans larva experiencing early-life stress (starvation or high pheromone) may enter the stress resistant, developmentally arrested dauer stage. When environmental conditions improve, larva will exit dauer and proceed with reproductive development (postdauers, PD). We have shown previously that hermaphrodites that experienced starvation-induced dauer (PDStv) exhibited a lower brood size compared to continuously fed animals (control, CON). The set of genes with the most significant differential expression between PDStv and CON adults have functions in fatty acid metabolism, and we found that double mutant combinations of the delta 9-desaturase genes, fat-5, fat-6, and fat-7, as well as mutations in their transcriptional regulators, nhr-49, sbp-1, and mdt-15, abrogated the decreased brood size phenotype. Using Oil Red O staining to examine levels of stored lipids, we found that PDStv adults displayed lower lipid stores in their intestines, but showed increased stored lipids in their embryos, when compared to CON adults. These results suggested that PDStv adults prioritize lipid production for reproduction rather than somatic maintenance after dauer exit. To test this hypothesis, we examined the brood sizes of PDStv adults that were cultivated on OP50 supplemented with different fatty acid molecules. We observed that supplementation with oleic acid (OA) resulted in a significant increase in brood size compared to animals fed only OP50, while other fatty acids resulted in no change or a decrease in brood size. We found that the OA-dependent increase in brood size required FAT-7, as well as the dafachronic acid (DA)-dependent activity of the DAF-12 steroid signaling pathway. Furthermore, we demonstrated that OA is required after dauer exit for the increased brood size phenotype. Together, these results suggest that OA may be acting as a signaling molecule to modulate PDStv reproductive plasticity through regulation of the DA-dependent DAF-12 pathway rather than acting in a nutritional capacity for reproduction.

Gautam Kao et al. (2006) European Worm Meeting "The ATPase ASNA-1 Positively Regulates Insulin Secretion in C. elegans and Mammals and Promotes Growth Non-autonomously"

Simon Tuck, Gautam Kao, Maria Still, Peter Naredia, Cecilia Nordensson, Agneta Rannlund

[European Worm Meeting, 2006]

Gautam Kao1, Cecilia Nordensson2, Maria Still2, Agneta Rnnlund1, Peter Naredi2 & Simon Tuck1. C. elegans embryos that hatch in the absence of food arrest growth during the first larval stage (L1). While much has been learned about the later diapause, called dauer that worms enter on encountering adverse conditions little is known presently about the mechanisms governing L1 arrest. We have identified and characterized a gene, asna-1, which when mutated, causes a complete but reversible arrest at the L1 stage even in the presence of food. We find that asna-1 encodes an ATPase that functions non-autonomously to regulate larval growth. asna-1 is expressed in a restricted set of sensory neurons and in the intestine; cells that produce insulin. asna-1 mutants have reduced insulin signaling while overexpression of asna-1 mimics the effects of overexpressing insulin. The human homologue ASNA1, which rescues the C. elegans mutant phenotype, is expressed at high levels in human pancreatic ?eta cells but not in other pancreatic endocrine or exocrine cell types. Increase or decrease of ASNA1 function in mouse insulinoma cells causes an increase or decrease in insulin secretion respectively. Similarly, reducing asna-1 gene function in C. elegans causes a decrease in levels of secreted insulin. We propose that ASNA1 is a novel evolutionarily conserved modulator of insulin signaling.

Zhao Y et al. (2007) BMC Genomics "Poly-G/poly-C tracts in the genomes of Caenorhabditis."

Zhao Y, O'Neil NJ, Rose AM

[BMC Genomics, 2007]

ABSTRACT: BACKGROUND: In the genome of Caenorhabditis elegans, homopolymeric poly-G/poly-C tracts (G/C tracts) exist at high frequency and are maintained by the activity of the DOG-1 protein. The frequency and distribution of G/C tracts in the genomes of C. elegans and the related nematode, C. briggsae were analyzed to investigate possible biological roles for G/C tracts. RESULTS: In C. elegans, G/C tracts are distributed along every chromosome in a non-random pattern. Most G/C tracts are within introns or are close to genes. Analysis of SAGE data showed that G/C tracts correlate with the levels of regional gene expression in C. elegans. G/C tracts are over-represented and dispersed across all chromosomes in another Caenorhabditis species, C. briggase. However, the positions and distribution of G/C tracts in C. briggsae differ from those in C. elegans. Furthermore, the C. briggsae dog-1 ortholog CBG19723 can rescue the mutator phenotype of C. elegans dog-1 mutants. CONCLUSIONS: The abundance and genomic distribution of G/C tracts in C. elegans, the effect of G/C tracts on regional transcription levels, and the lack of positional conservation of G/C tracts in C. briggsae suggest a role for G/C tracts in chromatin structure but not in the transcriptional regulation of specific genes.

Bhagwati P Gupta et al. (2002) West Coast Worm Meeting "Genetic analysis of the vulval mutants in C. briggsae"

Shahla Gharib, Bhagwati P Gupta, Paul W Sternberg, Jennifer X Li

[West Coast Worm Meeting, 2002]

To understand the evolution of developmental mechanisms, we are doing a comparative analysis of vulval patterning in C. elegans and C. briggsae. C. briggsae is closely related to C. elegans and has identical looking vulval morphology. However, recent studies have indicated subtle differences in the underlying mechanisms of development. The recent completion of C. briggsae genome sequence by the C. elegans Sequencing Consortium is extremely valuable in identifying the conserved genes between C. elegans and C. briggsae.

Antika TR et al. (2023) J Biol Chem "Sequence-specific targeting of C. elegans C-Ala to the D-loop of tRNAAla."

Horng JC, Hsu HL, Nazilah KR, Wang CC, Wang TL, Wang SC, Antika TR, Chuang TH, Chrestella DJ, Wang SW, Tseng YK, Pan HC

[J Biol Chem, 2023]

Alanyl-tRNA synthetase (AlaRS) retains a conserved prototype structure throughout its biology. Nevertheless, its C-terminal domain (C-Ala) is highly diverged and has been shown to play a role in either tRNA or DNA binding. Interestingly, we discovered that Caenorhabditis elegans cytoplasmic C-Ala (Ce-C-Ala_c) robustly binds both ligands. How Ce-C-Ala_c targets its cognate tRNA and whether a similar feature is conserved in its mitochondrial counterpart remain elusive. We show that the N- and C-terminal subdomains of Ce-C-Ala_c are responsible for DNA and tRNA binding, respectively. Ce-C-Ala_c specifically recognized the conserved invariant base G¹⁸ in the D-loop of tRNA^Ala through a highly conserved lysine residue, K934. Despite bearing little resemblance to other C-Ala domains, C. elegans mitochondrial C-Ala (Ce-C-Ala_m) robustly bound both tRNA^Ala and DNA and maintained targeting specificity for the D-loop of its cognate tRNA. This study uncovers the underlying mechanism of how C. elegans C-Ala specifically targets the D-loop of tRNA^Ala.