Hubbard EJA et al. (2019) Genetics "Biology of the Caenorhabditis elegans Germline Stem Cell System."

Schedl T, Hubbard EJA

[Genetics, 2019]

Stem cell systems regulate tissue development and maintenance. The germline stem cell system is essential for animal reproduction, controlling both the timing and number of progeny through its influence on gamete production. In this review, we first draw general comparisons to stem cell systems in other organisms, and then present our current understanding of the germline stem cell system in <i>Caenorhabditis elegans</i> In contrast to stereotypic somatic development and cell number stasis of adult somatic cells in <i>C. elegans</i>, the germline stem cell system has a variable division pattern, and the system differs between larval development, early adult peak reproduction and age-related decline. We discuss the cell and developmental biology of the stem cell system and the Notch regulated genetic network that controls the key decision between the stem cell fate and meiotic development, as it occurs under optimal laboratory conditions in adult and larval stages. We then discuss alterations of the stem cell system in response to environmental perturbations and aging. A recurring distinction is between processes that control stem cell fate and those that control cell cycle regulation. <i>C. elegans</i> is a powerful model for understanding germline stem cells and stem cell biology.

Jane Albert Hubbard E (2014) Methods "FLP/FRT and Cre/lox recombination technology in C. elegans."

Jane Albert Hubbard E

[Methods, 2014]

One of the most powerful aspects of biological inquiry using model organisms is the ability to control gene expression. A holy grail is both temporal and spatial control of the expression of specific gene products - that is, the ability to express or withhold the activity of genes or their products in specific cells at specific times. Ideally such a method would also regulate the precise levels of gene activity, and alterations would be reversible. The related goal of controlled or purposefully randomized expression of visible markers is also tremendously powerful. While not all of these feats have been accomplished in Caenorhabditis elegans to date, much progress has been made, and recent technologies put these goals within closer reach. Here, I present published examples of successful two-component site-specific recombination in C. elegans. These technologies are based on the principle of controlled intra-molecular excision or inversion of DNA sequences between defined sites, as driven by FLP or Cre recombinases. I discuss several prospects for future applications of this technology.

Hubbard EJA et al. (1996) Science "Evidence for physical and functional association between EMB-5 and LIN-12 in Caenorhabditis elegans."

Greenwald IS, Dong Q, Hubbard EJA

[Science, 1996]

The Caenorhabditis elegans LIN-12 and GLP-1 proteins are members of the LIN-12/Notch family of receptors for intercellular signals that specify cell fate. Evidence presented here suggests that the intracellular domains of LIN-12 and GLP-1 interact with the C. elegans EMB-5 protein and that the emb-5 gene functions in the same pathway as the lin-12 and glp-1 genes. EMB-5 is similar in sequence to a yeast protein that controls chromatin structure. Hence, a direct consequence of LIN-12 or GLP-1 activation may be an alteration of chromatin structure that produces changes in transcriptional activity.

Hubbard C et al. (2013) J Neurosci Res "Lanthionine ketimine ethyl ester partially rescues neurodevelopmental defects in unc-33 (DPYSL2/CRMP2) mutants."

Hubbard C, Baxter T, O'Brien S, Hardin T, Benda E, St John E, Holgado AM, Hensley K

[J Neurosci Res, 2013]

Lanthionine ketimine (LK) is a natural sulfur amino acid metabolite with potent neurotrophic activity. Proteomics indicate that LK interacts with collapsin response mediator protein-2 (CRMP2/DPYSL2/UNC-33), a brain-enriched protein that was shown to regulate cytoskeletal remodeling, neuronal morphology, and synaptic function. To elucidate further the molecular interplay and biological action of LK and UNC-33, we began examining the nervous system of Caenorhabditis elegans nematodes in which both LK concentrations and UNC-33 protein were manipulated. To this end, a cell-permeable LK-ester (LKE) was administered to developing C. elegans engineered to express yellow fluorescent protein (YFP) in cholinergic neurons (strain RM3128) or green fluorescent protein (GFP) in GABAergic neurons (strain CZ1200), and neural morphology was assessed. Fluorescent imaging analyses show that LKE exposure to wild-type animals induced neural commissure outgrowth, crossing over, and bundling in both neurites from GABAergic and cholinergic motor neurons. Additionally, when unc-33(e204) hypomorph mutant nematodes (D389N substitution mutants) were exposed to LKE, both the neuroanatomical defects of incomplete dorsoventral neural commissures and the ventral nerve cord gaps were partially rescued. In contrast, LKE did not rescue ventral nerve cord gaps found in unc-33(mn407) null mutant. Together these data suggest possible functions for LK as a regulator of neuritic elongation, corroborate roles for UNC-33/CRMP2 in the mechanism of LKE activity, and suggest the potential of LKE as a therapeutic molecule for neurological diseases involving CRMP2 dysfunction.

Hubbard EJA et al. (1997) Genes Dev "sel-10, a negative regulator of lin-12 activity in Caenorhabditis elegans, encodes a member of the CDC4 family of proteins."

Hubbard EJA, Greenwald IS, Kitajewski JK, Wu GY

[Genes Dev, 1997]

Mutations that influence lin-12 activity in Caenorhabditis elegans may identify conserved factors that regulate the activity of lin-12/Notch proteins. We describe genetic evidence indicating that sel-10 is a negative regulator of lin-12/Notch-mediated signaling in C. elegans. Sequence analysis shows that SEL-10 is a member of the CDC4 family of proteins and has a potential human ortholog. Coimmunoprecipitation data indicate that C. elegans SEL-10 complexes with LIN-12 and with murine Notch4. We propose that SEL-10 promotes the ubiquitin-mediated turnover of LIN-12/Notch proteins, and discuss potential roles for the regulation of lin-12/Notch activity by sel-10 in cell fate decisions and tumorigenesis.

Golden TR et al. (2008) Aging Cell "Age-related behaviors have distinct transcriptional profiles in Caenorhabditis elegans."

Dando C, Herren MA, Hubbard A, Melov S, Golden TR

[Aging Cell, 2008]

There has been a great deal of interest in identifying potential biomarkers of aging. Biomarkers of aging would be useful to predict potential vulnerabilities in an individual that may arise well before they are chronologically expected, due to idiosyncratic aging rates that occur between individuals. Prior attempts to identify biomarkers of aging have often relied on the comparisons of long-lived animals to a wild-type control. However, the effect of interventions in model systems that prolong lifespan (such as single gene mutations or caloric restriction) can sometimes be difficult to interpret due to the manipulation itself having multiple unforeseen consequences on physiology, unrelated to aging itself. The search for predictive biomarkers of aging therefore is problematic, and the identification of metrics that can be used to predict either physiological or chronological age would be of great value. One methodology that has been used to identify biomarkers for numerous pathologies is gene expression profiling. Here, we report whole-genome expression profiles of individual wild-type Caenorhabditis elegans covering the entire wild-type nematode lifespan. Individual nematodes were scored for either age-related behavioral phenotypes, or survival, and then subsequently associated with their respective gene expression profiles. This facilitated the identification of transcriptional profiles that were highly associated with either physiological or chronological age. Overall, our approach serves as a paradigm for identifying potential biomarkers of aging in higher organisms that can be repeatedly sampled throughout their lifespan.

Powolny AA et al. (2011) Exp Gerontol "The garlic constituent diallyl trisulfide increases the lifespan of C. elegans via skn-1 activation."

Singh SV, Powolny AA, Fisher AL, Hubbard A, Melov S

[Exp Gerontol, 2011]

Medicinal benefits of Allium vegetables, such as garlic, have been noted throughout recorded history, including protection against cancer and cardiovascular disease. We now demonstrate that garlic constituent diallyl trisulfide (DATS) increases longevity of Caenorhabditis elegans by affecting the skn-1 pathway. Treatment of worms with 5-10 M DATS increased worm mean lifespan even when treatment is started during young adulthood. To explore the mechanisms involved in the DATS-mediated increase in longevity, we treated daf-2, daf-16, and eat-2 mutants and found that DATS increased the lifespan of daf-2 and daf-16 mutants, but not the eat-2 mutants. Microarray experiments demonstrated that a number of genes regulated by oxidative stress and the skn-1 transcription factor were also changed by DATS treatment. Consistently, DATS treatment leads to the induction of the skn-1 target gene gst-4, and this induction was dependent on skn-1. We also found that the effects of DATS on worm lifespan depend on skn-1 activity in both in the intestine and ASI neurons. Together our data suggest that DATS is able to increase worm lifespan by enhancing the function of the pro-longevity transcription factor skn-1.

Robinson-Thiewes, Sarah et al. (2021) MicroPubl Biol

Robinson-Thiewes, Sarah, Kimble, Judith

[MicroPubl Biol, 2021]

Most eukaryotic genes make nascent transcripts that include both introns and exons. Introns are spliced out, usually co-transcriptionally, to generate mature mRNA (Herzel et al. 2017). Once considered a byproduct of transcription, introns are now known to affect virtually all aspects of mRNA metabolism, from transcriptional initiation to translation (Swinburne and Silver 2008; Chorev and Carmel 2012; Rose 2019). Genes can have multiple introns of variable size, but the first intron is often the longest (Smith 1988; Kriventseva and Gelfand 1999; Bradnam and Korf 2008). We wondered if the long first intron of a critical developmental regulator might affect its expression during development. To address this question, we analyzed expression of the C. elegans mpk-1b RNA, which encodes a nematode ERK/MAPK ortholog and has a long first intron (Figure 1A). Because mpk-1b is a germline-specific RNA (Lee et al. 2007; Robinson-Thiewes et al. 2020a), we assayed its expression along the developmental axis of the distal gonad (Figure 1B). Here, germ cells move through three regions as they progress from germline stem cells (GSCs) to differentiation: GSCs and GSC daughters primed for differentiation reside in the Progenitor Zone (PZ); germ cells enter meiotic prophase in the Transition Zone (TZ); and continue through meiotic prophase in the Early Pachytene (EP) region (Figure 1B)(Hubbard and Schedl 2019). Previously, we reported that a set of three ~1 kb deletions at distinct sites within the mpk-1b long intron did not affect its transcription or mRNA abundance (Robinson-Thiewes et al. 2020b). Here, we report effects of these deletions on production of the MPK-1B protein.

Ayuso, Cristina et al. (2019) MicroPubl Biol

Askjaer, Peter, Ayuso, Cristina

[MicroPubl Biol, 2019]

Several techniques are available for spatiotemporal control of genome recombination and gene expression in the nematode Caenorhabditis elegans. Here we report a novel tool to combine the powerful FLP-Frt and GAL4-UAS systems to increase their versality and to offer additional levels of control.FLP is an enzyme that catalyzes recombination between two short Frt DNA sequences and is frequently used to excise genomic fragments flanked by Frt sites, thereby either activating or knocking out gene expression, depending on the experimental design (Hubbard, 2014). Recently, we generated multiple strains that stably express FLP in different somatic tissues from single-copy transgenes and demonstrated that they in most cases induce recombination in ~100% of the cells of the expected tissue (Munoz-Jimenez et al., 2017). We subsequently constructed a strain for germline recombination to permanently knock out Frt-flanked genes or exons (Macas-Len and Askjaer, 2018).The GAL4-UAS system is based on the Saccharomyces cerevisiae Gal4p transcription factor and its cognate DNA target called upstream activating sequence (UAS). Typically, this bipartite system includes a series of driver strains expressing GAL4 in specific tissues and one or several strains with an effector gene downstream of UAS repeats. Wang and colleagues from the Sternberg laboratory recently optimized the GAL4-UAS system for C. elegans (cGAL) and reported several tissue-specific cGAL drivers (Wang et al., 2017). Moreover, they have developed a split cGAL toolkit where the DNA binding and activation domains are expressed as individual polypeptides, thereby enabling further fine-tuning of spatiotemporal control: only when and where the two components are co-expressed they will activate the UAS::effector transgene (Wang et al., 2018).

Lucanic M et al. (2013) Aging (Albany NY) "Age-related micro-RNA abundance in individual C. elegans."

Hubbard A, Graham J, Lucanic M, Benz CC, Melov S, Scott G, Lithgow GJ, Bhaumik D

[Aging (Albany NY), 2013]

Non-coding small RNAs of the micro-RNA class (miRNA) are conserved regulators of gene function with a broad impact on biological processes. We screened miRNA levels for age-related changes in individual worms and investigated their influence on the lifespan of the nematode C. elegans. We measured the abundance of 69 miRNAs expressed in individual animals at different ages with over thirty five thousand discrete quantitative nano-fluidic polymerase chain reactions. We found that miRNA abundance was highly variable between individual worms raised under identical conditions and that expression variability generally increased with age. To identify expression differences associated with either reproductive or somatic tissues, we analyzed wild type and mutants that lacked germlines. miRNAs from the mir-35-41 cluster increased in abundance with age in wild type animals, but were nearly absent from mutants lacking a germline, suggesting their age-related increase originates from the germline. Most miRNAs with age-dependent levels did not have a major effect on lifespan, as corresponding deletion mutants exhibited wild-type lifespans. The major exception to this was mir-71, which increased in abundance with age and was required for normal longevity. Our genetic characterization indicates that mir-71 acts at least partly in parallel to insulin/IGF like signals to influence lifespan.