Lee, Tong Young et al. (2015) International Worm Meeting "A simple 3D cultivation system to measure C. elegans fitness in a semi-natural simulated environment."

Lee, Jin Il, Lee, Tong Young

[International Worm Meeting, 2015]

Over the past forty years, C. elegans has been one of the most valuable laboratory model organisms for biological research. However, little is known about C. elegans in natural habitats. Cultivation conditions in the laboratory have limited C. elegans to growth in two dimensions with food availability plentiful and easy to locate. C. elegans in nature resides in soil, and rotten fruit and vegetative matter. These are complex three-dimensional environments that require worms to navigate in all directions to pinpoint sources of bacteria. We have developed a simple 3D NGM cultivation environment that allows C. elegans to move freely in a 3D agar matrix. OP50 E. coli forms distinct and sparse spherical colonies throughout the agar matrix rather than the familiar bacterial lawns that form on 2D plates. Wild-type worms grow and develop normally, and have similar sized brood size and growth rates as worms grown on the standard 2D NGM plates. On the other hand, sensory challenged mutants such as osm-6 mutants showed lowered brood sizes in the 3D environment compared to the 2D NGM plates. We surmise that osm-6 mutants have difficulty tracking and finding bacteria in 3D and this results in lowered fitness in the naturally simulated environment. We are conducting further experiments to see if our 3D cultivation system can be a useful correlative assay to test reproductive fitness of different genetic backgrounds or treatments.

Lee, Tong Young et al. (2017) International Worm Meeting "Observing the pattern of C. elegans egg-laying behaviors via 2D and 3D environment."

Lee, Tong Young, Yoon, Kyoung-hye, Lee, Jin Il

[International Worm Meeting, 2017]

The nematode C. elegans is one of the premier experimental model organisms today. In the laboratory, they display characteristic development, fertility, and behaviors in a 2D habitat. In nature, however, C. elegans is found in three dimensional environments such as rotting fruit. To investigate the biology of C. elegans in a 3D controlled environment, we designed a nematode cultivation habitat which we term the nematode growth tube or NGT-3D. Growth, brood size, and lifespan in NGT-3D are comparable with 2D NGM cultivation. Interestingly, we observe differences in patterns of feeding and egg-laying behaviors in 3D compared to 2D. We wonder if these differences affect the progeny in any way and are analyzing brood size and behavior patterns in the next generation. Additionally, we are screening through candidate mutants to identify any genes that are important for survival or fitness in 3D environments. Characteristic patterns of egg-laying and feeding behaviors observed in 3D might indicate a maternal behavior to benefit the survival and fitness of young in natural environments, and thus we plan to analyze the relationship between 3D egg-laying behaviors and another possible maternal behavior, internal hatching or worm bagging, that occurs in harsh environments. Overall, observing patterns of behaviors in a more natural 3D environment allows us to broaden our understanding of nematode biology and ecology

Lee, Tong Young et al. (2021) International Worm Meeting "Neuropeptides regulate a novel C. elegans oviposition behavior displayed in a three-dimensional environment"

Lee, Tong Young, Yoon, Kyoung-hye, Lee, Jin I.

[International Worm Meeting, 2021]

The C. elegans ecological niche is a complex and diverse three-dimensional environment found mostly in rotting fruit or soil compost. However, C. elegans behavior and physiology have mostly been studied in 2D culture in the lab. Previously, we designed a cultivation habitat which we term NGT-3D and NGB-3D in which growth, brood size, and lifespan in NGT-3D are comparable with 2D NGM plates. Using this 3D cultivation, we observed that the adult hermaphrodites spread the bacteria out from the center in a "nest" like pattern and remain at the edge of this nest. We then observed a novel stereotypical oviposition behavior in which the mother worms occasionally wander away from the bacterial nest, lay eggs, and then return to the bacteria. Using a candidate mutant analysis, we found that mutants defective in the neuropeptide PDF-1, as well as its cognate receptor PDFR-1 that regulates roaming behavior, were also defective in the novel 3D oviposition behavior laying eggs close to the bacteria. In addition, we identified mutants defective in the FMRF-like neuropeptide FLP-17 and its cognate receptor EGL-6 that together inhibit egg-laying were also defective in the novel 3D behavior. FLP-17 is expressed in the BAG sensory neuron, and the EGL-6 receptor expresses in the HSN neuron that regulates egg-laying. We showed that both egl-46 mutants that lack the BAG neuron as well as egl-1 mutants that lack the HSN neuron were also defective for the novel 3D oviposition behavior. To understand what cue may be causing mother worms to lay eggs away from OP50 in 3D, we found that 2D culture of C. elegans with a tiny "dot" colony of OP50 could elicit a similar egg-laying behavior in which the N2 mothers lay eggs far away from the bacteria. We found that low oxygen exacerbates the behavior, whereas high oxygen restores egg-laying in OP50. However, flp-17 mutants displayed defects in the oxygen-dependent egg-laying behavior. Finally, we tested whether defects in 3D oviposition behavior could induce decreased reproductive fitness in 3D and showed that flp-17 mutants have decreased brood sizes in 3D cultivation compared to 2D. Although low oxygen itself does not affect brood size, we found that cultivation with a high density of OP50 may be toxic to the young. Overall, we speculate that the novel 3D oviposition behavior may be a maternal behavior that increases the mothers' reproductive fitness.

Kalichamy, Saraswathi et al. (2015) International Worm Meeting "Hypergravity induces axon outgrowth defects in GABAergic motor neurons."

Lee, Tong Young, Lee, Jin Il, Kalichamy, Saraswathi

[International Worm Meeting, 2015]

Increased space travel and habitation are on the horizon with private companies competing to send people into space. However low gravity conditions are not without cost for the human body. Muscle and bone atrophy are a major problem especially for long-term travel and habitation in microgravity environments. Studies have mostly focused on gravity's effect on muscle to curb the problems of bone and muscle wasting. However the effect that gravity has on motor neuron development and axonal outgrowth and pathfinding has been overlooked. Using C. elegans as a model for motor neuron development, we decided to look at the effect that hypergravity may have on axonal outgrowth and pathfinding in the D-type GABAergic motor neurons. We first designed and tested a worm hypergravity cultivation system in which C. elegans can be exposed to 100G force gravity for several days. Next, we used an unc-25::GFP strain that expresses GFP in the D-type motorneurons. The axon commissural projections run along the antero-posterior and ventrodorsal direction in response to guidance molecules. We investigated the trajectory of projections in hypergravity conditions by subjecting synchronized unc-25::GFP eggs to 100G centrifugation for 3 days. At the end of the 3rd day, we analysed the worms and found branched and bent commissures that often moved away from the target instead of running towards the dorsal cord. To determine if there is a critical period of hypergravity's effect on projections, we exposed the worms to centrifugation for 2, 2.5 and 3 days. Our preliminary data indicates that commissures showed increased branching, bending and failure to reach the dorsal cord after 2.5/3 days of hypergravity exposure. Interestingly, most of the commissural defects occurred among the mid-body commissures, and we rarely observed defects at the most anterior or posterior commissures. We are currently performing genetic experiments to identify how the axonal defects arise from hypergravity.

Abate JP et al. (2009) Cell Metab "Life is short, if sweet."

Blackwell TK, Abate JP

[Cell Metab, 2009]

Insulin is essential for glucose homeostasis, but reducing its activity delays the aging process in model organisms. In this issue of Cell Metabolism, Lee et al. (2009) show how these effects of insulin signaling intersect when glucose is fed to C. elegans.

Lee C et al. (2017) Bio Protoc "Single-molecule RNA Fluorescence in situ Hybridization (smFISH) in Caenorhabditis elegans."

Sorensen EB, Lee C, Seidel HS, Lynch TR, Crittenden SL, Kimble J

[Bio Protoc, 2017]

Single-molecule RNA fluorescence <i>in situ</i> hybridization (smFISH) is a technique to visualize individual RNA molecules using multiple fluorescently-labeled oligonucleotide probes specific to the target RNA ( Raj <i>et al.</i>, 2008 ; Lee <i>et al.</i>, 2016a ). We adapted this technique to visualize RNAs in the <i>C. elegans</i> whole adult worm or its germline, which enabled simultaneous recording of nascent transcripts at active transcription sites and mature mRNAs in the cytoplasm ( Lee <i>et al.</i>, 2013 and 2016b). Here we describe each step of the smFISH procedure, reagents, and microscope settings optimized for <i>C. elegans</i> extruded gonads.

Hogger CH et al. (1977) Nematologica "Surface scanning observations of changes in C. briggsae during aging."

Estey RH, Zuckerman BM, Hogger CH, Kisiel MJ

[Nematologica, 1977]

SEM observations of adult Caenorhabditis briggsae females showed differences between young and old nematodes. In young nematodes the cuticle was generally smooth, whereas in old ones it was wrinkled. Deirids were located at the level of the excretory pore in the lateral field. They were distinct in young nematodes but indistinct in old ones. The oral opening was formed by six lips, which were closed in old nematodes and open in young ones. The vulva possessed two semi-circular lips and was bordered by two lateral flaps. These lips were smooth in young specimens and wrinkled in old ones. Cryofractures of old nematodes showed cavities in the intestinal epithelium corresponding to areas in which age pigment granules normally occur. No such cavities were seen in young nematodes.

Dawes-Hoang RE et al. (2003) Dev Cell "Bringing classical embryology to C elegans gastrulation."

Zallen JA, Wieschaus EF, Dawes-Hoang RE

[Dev Cell, 2003]

In a recent paper, Lee and Goldstein develop an explant assay that recapitulates key aspects of gastrulation in C. elegans and permits classical embryological manipulations. The resulting detailed analysis of cell behavior will ultimately extend to broader issues, such as, whether morphogenesis can be described as the sum of single-cell events or if unique phenomena emerge at the multicellular level.

Sutphin GL (2019) Elife "A new defense in the battle of the sexes."

Sutphin GL

[Elife, 2019]

Young <i>Caenorhabditis elegans</i> hermaphrodites use their own sperm to protect against the negative consequences of mating.

Bauer, Jack et al. (2021) MicroPubl Biol

Labb, Jean-Claude, Lacroix, La, Bauer, Jack

[MicroPubl Biol, 2021]

To perturb actomyosin function in the primordial germ line, we first monitored germ line organization in L1-stage animals bearing temperature-sensitive (ts) alleles in genes encoding actomyosin regulators and that were reported to interfere with cytokinesis during embryogenesis (Davies et al. 2014). Previous work demonstrated that the initial stages of germline expansion occur normally in cyk-4(ts) and zen-4(ts) animals raised at restrictive temperature from the L1 stage (Lee et al. 2018). We found that primordial germ line organization in cyk-1(ts), nmy-2(ts), cyk-4(ts) or zen-4(ts) L1 larvae maintained at restrictive temperature for 12h was no different than control (Figure 1A-B). Furthermore, the first primordial germ cell (PGC) division occurred normally upon feeding these animals at restrictive temperature with typical bacterial food (E. coli OP50). As noted previously (Lee et al. 2018), germ line disorganization and sterility were observed in all cases when animals reached adulthood (Figure 1B).