Driesschaert, Brecht et al. (2022) MicroPubl Biol

Driesschaert, Brecht, Temmerman, Liesbet

[MicroPubl Biol, 2022]

The Q system is a genetic tool developed to deliver spatiotemporal control over gene expression (Giles et al. 1991; Potter et al. 2010; Wei et al. 2012). Although it has already been adapted for use in C. elegans by Wei et al. in 2012, to date, the Q system has not been applied extensively in this nematode. In the relatively few available reports, it is mainly used to constitutively restrict gene expression in a spatial manner (e.g. Schild et al. 2014; Schild and Glauser 2015; Jee et al. 2016; Tolstenkov et al. 2018; Chiyoda et al. 2021), while but a handful of studies also explore the temporal aspect of the system (Matus et al. 2015; Yuan et al. 2016; Cottee et al. 2017; Hoang and Miller 2017). We aimed to apply this tool in the C. elegans nervous system to gain both spatial and temporal control over expression of a gene encoding a reporter protein that is targeted to the secretory pathway. Despite our efforts, we here report that in our hands, the Q system is not suitable for application in the neurons due to a lack of dynamic range.

Huang CG et al. (2006) Mol Biotechnol "Isolation of C. elegans deletion mutants following ENU mutagenesis and thermostable restriction enzyme PCR screening."

Lamitina T, Agre P, Strange K, Huang CG

[Mol Biotechnol, 2006]

The ability to generate null mutants is essential for studying gene function. Gene knockouts in Caenorhabditis elegans can be generated in a high throughput manner using chemical mutagenesis followed by polymerase chain reaction (PCR) assays to detect deletions in a gene of interest. However, current methods for identifying deletions are time and labor intensive and are unable to efficiently detect small deletions. In this study, we expanded the method pioneered by Wei et al., which used the thermostable restriction enzyme PspGI and tested the usefulness of other thermostable restriction enzymes including BstUI, Tsp45I, ApeKI, and TfiI. We designed primers to flank one or multiple thermostable restriction enzymes sites in the genes of interest. The use of multiple enzymes and the optimization of PCR primer design enabled us to isolate deletion in 66.7% of the genes screened. The size of the deletions varied from 330 bp to 1 kb. This method should make it possible for small academic laboratories to rapidly isolate deletions in their genes of interest.

Thurman, Michellie et al. (2021) MicroPubl Biol

Sun, Haonan, Praitis, Vida, Kubica, Sam, Thurman, Michellie

[MicroPubl Biol, 2021]

Calcium signaling is known to play a critical role in cell migration (Ridley et al., 2003; Wei et al., 2012). In C. elegans, embryos with disruptions in the secretory pathway calcium ATPase gene pmr-1 show defects in cell migration that result in lethal phenotypes. Previous work has shown that pmr-1(lof) phenotypes can be suppressed by changes in the activity of the calcium channels IP3-receptor/ITR-1 and ryanodine receptor/UNC-68, indicating that cell migration defects are the results of changes in calcium homeostasis (Praitis et al., 2013). To identify additional genes important for cell migration during embryogenesis, we performed a genetic screen to isolate additional suppressors of the pmr-1(ru5) strain, reasoning that disruption of cell migration due to changes in pmr-1 activity could be suppressed by commensurate changes in other genes that regulate calcium levels or signaling. In this screen, we identified the kez8 allele. We confirmed that the kez8 allele suppresses the pmr-1(ru5) mutant phenotype by examining the percentage of viable progeny produced by pmr-1(ru5); kez8 strains when grown under restrictive conditions. The viability of the pmr-1(ru5); kez8 strain is 67%, significantly higher than the 3.9% viability observed in the pmr-1(ru5) control strain at 25C (Table 1; p<0.01).

Song BM et al. (2012) J Neurosci "Serotonin activates overall feeding by activating two separate neural pathways in Caenorhabditis elegans."

Song BM, Avery L

[J Neurosci, 2012]

Food intake in the nematode Caenorhabditis elegans requires two distinct feeding motions, pharyngeal pumping and isthmus peristalsis. Bacteria, the natural food of C. elegans, activate both feeding motions (Croll, 1978; Horvitz et al., 1982; Chiang et al., 2006). The mechanisms by which bacteria activate the feeding motions are largely unknown. To understand the process, we studied how serotonin, an endogenous pharyngeal pumping activator whose action is triggered by bacteria, activates feeding motions. Here, we show that serotonin, like bacteria, activates overall feeding by activating isthmus peristalsis as well as pharyngeal pumping. During active feeding, the frequencies and the timing of onset of the two motions were distinct, but each isthmus peristalsis was coupled to the preceding pump. We found that serotonin activates the two feeding motions mainly by activating two separate neural pathways in response to bacteria. For activating pumping, the SER-7 serotonin receptor in the MC motor neurons in the feeding organ activated cholinergic transmission from MC to the pharyngeal muscles by activating the Gs signaling pathway. For activating isthmus peristalsis, SER-7 in the M4 (and possibly M2) motor neuron in the feeding organ activated the G(12) signaling pathway in a cell-autonomous manner, which presumably activates neurotransmission from M4 to the pharyngeal muscles. Based on our results and previous calcium imaging of pharyngeal muscles (Shimozono et al., 2004), we propose a model that explains how the two feeding motions are separately regulated yet coupled. The feeding organ may have evolved this way to support efficient feeding.

Von Stetina SE et al. (2015) Dev Biol "PAR-6, but not E-cadherin and -integrin, is necessary for epithelial polarization in C. elegans."

Von Stetina SE, Mango SE

[Dev Biol, 2015]

Cell polarity is a fundamental characteristic of epithelial cells. Classical cell biological studies have suggested that establishment and orientation of polarized epithelia depend on outside-in cues that derive from interactions with either neighboring cells or the substratum (Akhtar and Streuli, 2013; Chen and Zhang, 2013; Chung and Andrew, 2008; McNeill et al., 1990; Nejsum and Nelson, 2007; Nelson et al., 2013; Ojakian and Schwimmer, 1994; Wang et al., 1990; Yu et al., 2005). This paradigm has been challenged by examples of epithelia generated in the absence of molecules that mediate cell-cell or cell-matrix interactions, notably E-cadherin and integrins (Baas et al., 2004; Choi et al., 2013; Costa et al., 1998; Harris and Peifer, 2004; Raich et al., 1999; Roote and Zusman, 1995; Vestweber et al., 1985; Williams and Waterston, 1994; Wu et al., 2009). Here we explore an alternative hypothesis, that cadherins and integrins function redundantly to substitute for one another during epithelium formation (Martinez-Rico et al., 2010; Ojakian et al., 2001; Rudkouskaya et al., 2014; Weber et al., 2011). We use C. elegans, which possesses a single E-cadherin (Costa et al., 1998; Hardin et al., 2013; Tepass, 1999) and a single -integrin (Gettner et al., 1995; Lee et al., 2001), and analyze the arcade cells, which generate an epithelium late in embryogenesis (Portereiko and Mango, 2001; Portereiko et al., 2004), after most maternal factors are depleted. Loss of E-cadherin(HMR-1) in combination with -integrin(PAT-3) had no impact on the onset or formation of the arcade cell epithelium, nor the epidermis or digestive tract. Moreover, B-integrin(PAT-3) was not enriched at the basal surface of the arcades, and the candidate PAT-3 binding partner -laminin(LAM-1) was not detected until after arcade cell polarity was established and exhibited no obvious polarity defect when mutated. Instead, the polarity protein par-6 (Chen and Zhang, 2013; Watts et al., 1996) was required to polarize the arcade cells, and par-6 mutants exhibited mislocalized or absent apical and junctional proteins. We conclude that the arcade cell epithelium polarizes by a PAR-6-mediated pathway that is independent of E-cadherin, -integrin and -laminin.

Ellington, Clayton T. et al. (2020) MicroPubl Biol

Hayden, Andrew J., Goodman, Miriam B., Luccioni, Marina D., Ramlan, Lauren I.E., Guha, Sujay, O'Connell, Lauren A., Osman, Mohammed A.M., Ellington, Clayton T., LaGrange, Zack B., Vogt, Miranda A.

[MicroPubl Biol, 2020]

Plants synthesize many structurally diverse organic molecules to interact with the organisms around them. These compounds can act at the receptor level in target organisms to aid the plant in defense or to facilitate a symbiotic relationship (Bahar et al., 2007; Wink, 2018). We have not exhausted these plant-derived secondary metabolites as a source of novel drug therapeutics for pressing human health challenges because screening them for potential function as neuronal actuators is difficult. Here, we explore (1) using C. elegans as an efficient and practical organism for screening plant compounds for potential bioactivity in the nervous system, and (2) using these methods in an undergraduate classroom to bring real scientific discoveries to teaching laboratories. C. elegans are well studied, have essential genetic tools, short generation times, and easy culture techniques (Corsi et al., 2015; OReilly et al., 2014). Moreover, C. elegans navigates its environment by relying on chemosensation, triggering chemotaxis in response to odorants (Bargmann, 2006; Margie et al., 2013). These characteristics make it a potentially effective organism for screening phytochemicals and teaching in an undergraduate laboratory setting.We first screened five commercially available plant compounds for potential actuators of neuronal function using C. elegans chemotaxis behavior (Figure 1A), including (S)-(+)-carvone, eugenol, cis-jasmone, linalool, and ()-ɑ-thujone (Figure 1B). The chemotaxis response in the N2 wild type strain differed across plant compounds and solvent control (ANOVA, F(5)=10.52, p=7.5910-5). In particular, (S)-(+)-carvone elicited a strong repulsion effect compared to solvent (p=0.0004) and all other compounds ((S)-(+)-carvone versus: eugenol p=0.005, cis-jasmone p=0.003, linalool p=0.008, ()-ɑ-thujone p=0.00009). No other compounds significantly differed between each other or solvent control at the concentration that was tested (20 mM), which is surprising as these compounds have been previously shown to produce interesting neuromodulatory and behavioral effects in other animals: cis-jasmone repels aphids (Birkett et al., 2000), ()-ɑ-thujone is a GABAA receptor antagonist (Hld et al., 2000), and both linalool (Batista et al., 2008) and eugenol (Chung et al., 2014) alleviate glutamate-induced nociceptive pain. It may be that dose-response curves or different genetic strains would be required to fully explore the effects of these compounds in nematodes. Regardless, to our knowledge, this is the first demonstration of carvone as a repellent in C. elegans and we conducted follow-up experiments to gain insight into potential mechanisms.We next tested whether there is genetic variation in chemotaxis response elicited by plant compounds by comparing chemotaxis behavior across divergent C. elegans strains (Figure 1C). Such variation between strains could allow for genome wide association studies to identify potential neuronal substrates (Cook et al., 2017). We tested (S)-(+)-carvone as well as ()-ɑ-thujone due to its known interactions with GABAA receptors (Olsen, 2000). Across four wild isolate strains and the N2 wild type strain, there was a significant effect of compound, but not strain, on chemotaxis behavior (2-way ANOVA, Compound: F(2)=4.679, p=0.0136; Strain: F(4)=1.821, p=0.1392). (S)-(+)-carvone elicited a negative chemotaxis response compared to both solvent (p=0.0409) and ()-ɑ-thujone (p=0.0201). The chemotaxis behavior elicited by ()-ɑ-thujone did not significantly differ from solvent (p=0.9444), mirroring results from the first screening experiment. These findings indicate that screening the number of strains and replicates required for genome wide association mapping is outside the scope of a single small undergraduate laboratory course, but leaves open the possibility that screening additional wild isolates could be valuable.

Ashley, Nicholas et al. (2019) MicroPubl Biol

Ashley, Nicholas, Holgado, Andrea M.

[MicroPubl Biol, 2019]

Aging can be defined by many different physiological pathways that emphasize changes in a variety of cells and tissues. Disease and death are often results of aging (Via et al., 2013). However, specific mechanisms in different cell types offer multiple hypotheses regarding the definition of aging (Via et al., 2007). Although some researchers target specific aspects of physiological processes as major contributors to aging, such as free radical theory (Harman, 2003), inflammation theory (Chung et al., 2001), mitochondrial theory (Cadenas and Davies, 2000; Sastre et al., 2000), immunologic theory (Franceschi et al., 2000), current hypotheses suggest that aging results from alterations of multiple systems working at the same time (Weinert and Timiras, 2003). Central Nervous System neural networks have been identified as particularly important for understanding cellular processes that cause aging, which is partly characterized by cell death (Yonekawa and Thorburn, 2013). Macroautophagy (hereon referred as Autophagy) is a cellular housekeeping mechanism that uses a double membrane to target and engulf cell products forthe formation of autophagosomes. These double membrane organelles then fuse to lysosomes where cell products aredegraded and recycled (Nakamura and Yoshimori, 2018). Reports show that autophagy plays an important role in pathogen defense, development, starvation adaptations, and aging (Mizushima et al., 2008).Analysis of autophagy mutants in C. elegansrevealed that bec-1/Atg6/Beclin 1is essential for dauer development, a quiescent state that survives harsh conditions such as lack of nutrients, high nematode density, and high temperatures by inducing autophagy (Melndez et al., 2003; Melndez and Levine 2009). To further investigate the phenotypes associated with the bec-1(ok691)mutation, we studied nematodes possessing a 3000 base pair deletion mutation (allele ok691) in the bec-1locus (Figure 1A). Previous reports show that the bec-1(ok691)mutation is lethal, however a small proportion of homozygous bec-1(ok691)animals reach adulthood due to maternal effect, but do not reproduce due to sterility (Melendez and Levine 2009). Our analysis of survival throughout adulthood shows thatlifespan is significantly reduced in homozygous bec-1(ok691)mutants (Ashley and Holgado, 2019a). In contrast, heterozygous bec-1(ok691)mutant lifespan is not significantly different from control animals, ruling out previously discovered haploid insufficiency effects of Beclin 1 on autophagy in C. elegans (Sinha and Levine, 2008). Previous research has found increased lifespans of daf-2mutant C. elegans, a genetic lesion that affects the insulin-like signaling pathway and promotes constitutive dauer formation (Kimura et al. 1997). Furthermore, this daf-2-dependent extension of lifespan was eliminated in bec-1(ok691); daf-2(e1370)double mutants. Together, these findings support the notion that the autophagy gene product BEC-1 is needed for the daf-2-dependent lifespan extension of C. elegans. Additionally, double mutantsAtg-7and bec-1(ok691)also show a decrease in lifespan in nematodes with inherent dietary restriction (Tth et al., 2008). The loss of autophagy function in the motor cortex has been associated with progression of neurodegenerative symptoms in Parkinsons disease (Kaila and Lang 2015; Fahn et al. 2004). We showed locomotion in C. elegansmutants for bec-1had significant defects (Ashley and Holgado, 2019b). These defects in locomotion suggest a role of autophagy controlling behaviors that are neuronal dependent. Considering our findings of defective locomotion and lifespan in abec-1(ok691)mutant background, we proceeded our research by assessing density of motor neurons in the ventral nerve cord of C. elegans. After analyzing possible effects of the bec-1(ok691)mutation on neuronal density, we followed the transgene juIs76as it produced GFP marked D-type motor neurons (Figure 1 B). These studies showed a delay in development of ventral D-type motor neurons in bec-1(ok691)homozygous mutants (Figure 1 C). Maturation and development of VD motor neurons to the levels of controls were seen on day 5 of adulthood in bec-1(ok691)homozygous mutants (Ashley and Holgado, 2019c). Previous research has found lineage timing of GABAergic (VD) motor neuron differentiation in C. elegansto occur before animals reach adulthood (Jin et al., 1994). However, our discovery in delayed lineage timing of VD motor neurons suggests a potential role of BEC-1 in neurodevelopment. This is consistent with findings in mouse models, where ortholog Beclin 1plays an essential role in cell differentiation during development (Cecconi and Levine, 2008). Instead of observing rapid neurodegeneration of VD motor neurons, resulting from the bec-1(ok691)mutation, we observed a rapid decrease in lifespan as VD motor neurons were differentiated. There is additional evidence that suggests autophagys role in mechanisms of cell editing in early developmental stages of C. elegans(Di Bartolomeo et al., 2010). These conclusions should be considered as preliminary as we havenot verified by an alternative line of investigation (e.g., a secondallele or transgene rescue) that the observed phenotypes are specific to bec-1(ok691).Specific mechanisms in which neurodegeneration act are still unknown. Future investigations observing the status of autophagy in bec-1(ok691)mutants may contribute novel knowledge into suggesting how autophagy may influence a delay in development of VD motor neurons or uncover pieces of the mechanism causing neurodegeneration in similar autophagy mutants.