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Hegarty, E., Martinez, M.A., Mondal, S., Matus, D.Q., Adikes, R.C., Smith, J.J., Ben-Yakar, A.
[
International Worm Meeting,
2019]
Recent advances in the generation of knock-in alleles by CRISPR/Cas9 and the use of small degron tags, such as the auxin-inducible degron (AID) to conditionally deplete proteins of interest, is revolutionizing modern C. elegans cell biology. We are interested in optimizing and combining these new technologies with high-resolution, high-throughput imaging. We present here preliminary results combining CRISPR/Cas9 and AID technologies with a microfluidic platform housed on our laboratory's Yokogawa spinning disk confocal. The previously published AID system in C. elegans requires the use of the natural auxin indole-3-acetic-acid (IAA), which is insoluble in water and inhibits E. coli OP50 growth. We have improved upon the delivery of the AID system by incorporating the use of a synthetic analog of auxin, 1-naphthaleneactic acid (NAA), that is water-soluble, conducive to bacterial growth, and compatible with M9 buffer. To pair this protein depletion system with high-resolution, high-content imaging, we are also piloting a microfluidic system, the vivoChip, for the imaging of the third larval stage of C. elegans development. During the L3 stage, many morphogenetic events occur, including uterine and vulval development. We are using the vivoChip to immobilize L3 larvae and visualize uterine-vulval attachment, where the somatic anchor cell (AC) breaches the underlying basement membrane, initiating uterine-vulval attachment, and establishing the nematode reproductive tract. We present here results of pairing the L3 microfluidics device with spinning disk confocal imaging of AC invasion and vulval morphogenesis, to investigate whether reduced anesthetics reveal any changes to the dynamics of normal uterine-vulval development.
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[
International Worm Meeting,
2013]
H3K79 methylation has been linked to transcriptional activation however the mechanism of H3K79 targeting and function are not well understood. H3K79 methylation is carried out by the Dot1 family of histone methyltransferases, which are characterized by a class I SAM-dependent methyltransferase domain. In all studied organisms, DOT1 is solely responsible for all H3K79 methylation states as the knockout of this gene results in a total loss of mono- di- and tri- methylation of H3K79 methylation. In yeast, non-methylated H3K79 is found at silenced telomeres, whereas H3K79 methylation at telomeres disrupts transcriptional silencing. In humans, leukaemia causing MLL fusions to AF9 recruit Dot1, and inactivation of Dot1 reduces expression of MLL-AF9 targets and inhibits progression of the disease. Dot1 has also been implicated in DNA damage checkpoint control and repair, the cell cycle, and is required for early embryonic development in both mice and flies. We are carrying out a functional analysis of H3K79 methylation in C. elegans. The worm genome has six DOT1L homologs, of which only Y39G10AR.18 is highly expressed across all developmental stages. As in other organisms, H3K79me2 and H3K79me3 are present on the bodies of transcriptionally active genes in early embryos and L3 larvae (Liu et al 2011). However, the pattern of H3K79me1 differs in early embryos and L3 larvae. Regions of high H3K79me1 in early embryos often lack this modification in L3 larvae. We are assessing which genes carry out H3K79 methylation and studying the function and developmental regulation of H3K79 methylation.
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[
International C. elegans Meeting,
1995]
The dauer formation pathway requires the animal to assimilate environmental inputs (food, pher- omone, and temperature) and regulate its dev- elopment (as a dauer or an L3 larvae) accordingly. We are cloning the gene
daf-23, a maternal effect dauer constitutive gene which acts in the
daf-23/
daf-2/daf-16/daf-18 branch of the dauer genetic epistasis pathway. This branch has dramatic effects on senescence as well as on dauer form- ation. Unlike the other branches of the dauer pathway, this branch has no known pleiotropies, suggesting that it acts downstream of the sensory neurons, perhaps in secretory neurons or in down- stream tissues.
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[
East Coast Worm Meeting,
2004]
We are interested in studying organ development and function in order to understand both the normal processes and how improper organ development and function can lead to birth defects or cancer. The research in this project focuses on how an organ's structure, function, and response can be modified by external stimuli, such as environmental conditions. The C. elegans excretory system is a good model for organ development and function because of its simplicity. The excretory system is composed of only four cell types, yet it performs all of the necessary functions of osmotic regulation and waste removal, similar to mammalian kidneys. The simple structure of the excretory system makes it possible to study development and function at the cellular level. Dauer formation is an example of a change induced by environmental conditions in C. elegans . During dauer formation, the morphology of numerous cells and organs is altered, including the pharynx, hypodermis, and excretory system (Riddle, Blumenthal, Meyer, and Priess, 1997). It was previously shown that the excretory system might play a role in the C. elegans ' morphological transition into dauer state (Nelson and Riddle, 1984). We are following up on this research using GFP markers to help visualize each component of the excretory system. We currently have markers for the excretory cell and excretory duct and are working on markers for the excretory pore and gland cells. Using the markers, we are able to observe the morphology of the excretory system during its development. As an initial experiment, we compared the morphology of the excretory duct in normal L3 larvae and L3 larvae that had entered dauer. Our studies showed there is a difference in the excretory duct morphology between dauer and non-dauer larvae. In dauer larvae, the duct's position had shifted, the duct cell body was elongated, and the appendages were hyper-extended as compared to normal L3 larvae. We plan to expand these studies to other excretory cell types, and to investigate how these changes alter the excretory system function. Riddle, D.L., Blumenthal, T., Meyer, B.J., and Priess, J.R., C. elegans II (1997): 739-768 Nelson, F.K and Riddle, D.L., J. Exp. Zool. (1984) Jul; 231 (1): 45-56
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[
International Worm Meeting,
2015]
How does the nervous sytem change during development to enable mature behavior? We use the development of chemotaxis behavior as model to study maturation and have shown that L3 larvae respond worse than adults in attractive diacetyl chemotaxis assays. However, L3 larvae are not generally bad at chemotaxis, since they respond similarly to adults in repulsive nonanone chemotaxis assays. To begin to identify neural mechanisms underlying maturation, we mapped both the adult and larval sensory circuits required for attractive diacetyl chemotaxis. Based on behavioral analysis of sensory neuron mutants, we found that the adult sensory circuit contains more neurons than the L3 circuit, suggesting that changing circuit composition may play a crucial role in behavioral maturation. To further characterize differences between the adult and L3 cellular circuits we examined the activity of individual sensory neurons from each circuit. While a microfluidic device for recording neuronal activity from adult worms is published, none had existed for L3 worms. In collaboration with N. Chronis and D. Bazopoulou we developed a novel microfluidic device that traps L3 animals and enables ready imaging of individual neurons. We previously reported that in both adult and L3 worms AWA sensory neurons respond to the addition of diacetyl. Interestingly, our current results show that L3 AWA responses are less reliable than adult responses at a lower diacetyl concentration. We also find, in support of our behavioral results, that different sets of sensory neurons encode diacetyl in adult and L3 animals. Moreover, the functional adult circuit is larger than the L3 one. Collectively, our results show that for adult and L3 worms different cellular circuits are required for attractive chemotaxis behavior, different sensory neurons encode diacetyl, and odors are encoded by cells which are dispensable for behavior. We will present these findings and our results analyzing differences in molecular pathways between L3 and adults.
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[
C. elegans: Development and Gene Expression, EMBL, Heidelberg, Germany,
2010]
In this study, we investigated the locomotion behavior changes at different developmental stages in Caenorhabditis elegans exposed to metals for 4h. No obvious differences could be observed in young adults exposed to examined metals, and only exposure to 100 mM of examined metals could signi?cantly decrease the locomotion behaviors of L4 larvae. In contrast, exposure to 50 and 100 mM of examined metals induced noticeable repression of locomotion behaviors at L1L3 larval stages, and a signi?cant decrease of locomotion behaviors could be observed in L1 larvae exposed to Pb and Hg, and in L2 larvae exposed to Hg at the concentration of 2.5 mM. Moreover, the L1-, L2-, and L3- larvae exposed to metals for 4h exhibited similar neuro behavioral toxicity manner to L4- larvae exposed to metals for 24h. Therefore, younger larvae showed more severe de?cits in neuro behavioral phenotypes than L 4 larvae and young adults in metal-exposed nematodes.
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[
International Worm Meeting,
2015]
C. elegans life history is dependent on environmental cues transduced through several signaling pathways. In favorable environmental conditions C. elegans develops continuously through four larval stages before molting into the adult stage. In contrast, adverse environmental conditions promote entry into the stress resistant dauer larva stage immediately following the second larval molt. Dauer larvae possess distinct properties including cellular and developmental arrest, extended lifespan, and discontinued feeding. If environmental conditions improve, dauer larvae recover to post-dauer L3 larvae, which are developmentally identical to continuously developing L3 larvae. Studies of dauer larvae have seeded discoveries in diverse fields, ranging from aging to neurobiology. Many of these studies rely on the unambiguous identification of dauer larvae. The most useful method to identify dauer larvae would be simple, scalable, and feasible even in mutant backgrounds that are defective in some aspects of dauer morphogenesis. None of the current methods possess all of these qualities. Here, we describe such a method taking advantage of the inability of dauer larvae to feed. Fluorescent beads are added to the bacterial food source, and dauer larvae are identified by a lack of beads within their digestive tract. We describe how this assay can be used to isolate dauer larvae formed by any of three common methods: starved plates, exogenous pheromone, and dauer-constitutive mutations. Lack of beads correlates well with other known markers of dauer formation, including greatly reduced pumping rate, presence of dauer alae and SDS resistance. We find that using beads rather than SDS-resistance to identify dauer larvae enables the recovery of SDS-sensitive mutants, including cuticle mutants and certain mutants within the dauer formation pathway. Finally, lack of beads also identifies molting larvae, extending the utility of this assay beyond dauer larvae.
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[
International C. elegans Meeting,
1995]
The C .elegans genome sequencing initiatives have identified many genes which have no similarity to other database sequences. Given the deep evolutionary separation between nematodes and other model organisms we suspect that many of these will be nematode-specific. We have initiated an est project on the spirurid parasite B.=A0malayi (a causative agent of human lymphatic filariasis): we aim to identify genes important in the development of infective L3 larvae in the invertebrate/vertebrate host transition. A full length library was made in the lambda ZapII vector from SL-oligo(dT) cDNA from infective L3. So far 300 clones have been 5' tag sequenced. These can be grouped into 4 classes: (1) Clones unique to our dataset; (2) universal housekeeping genes; (3) homologues of functionally identified nematode genes (both C. elegans and parasites) and (4) clear homolgues of otherwise anonymous C. elegans ORFs and DNAs. Classes (3) and (4) constitute a possible "nematode-specific" group and the availability of such phylogenetically distant homologues should aid both parasitic and freeliving research communities.
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[
Development & Evolution Meeting,
2006]
With their remarkable potential to self-renew and develop into many different cell types in the body, stem cells have been at center stage both in developmental biology and regenerative medicine. The study of what makes a cell stem cell will not only help us understand normal cell development, but also promises powerful therapeutic potential for a variety of human diseases. However, the molecular mechanisms of stem cell regulation are still poorly understood. This approach took advantage of a gene-expression profiling that used DNA microarray technology to identify genes preferentially expressed in the germline stem cells of C. elegans, which may play an important role in the regulation of germline stem cell function. We defined a subset of forty genes that reproducibly gave sterile phenotypes upon functional depletion by RNAi screen of L3 larvae (soaking) and further designed a screen to identify genes for which inhibition by RNAi using microinjection of dsRNA elicited defects in germline stem cell development and function. We obtained strong germline phenotype for most of the genes tested, indicating that these factors are required for normal germline proliferation and development. The long term goal of current research is to determine the gene expression networks that define stem cell characteristics.
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[
International Worm Meeting,
2015]
Eukaryotic genomes are organized into domains of differential structure and activity. Through chromatin state mapping using 17 chromatin marks in L3 larvae, we identify 20 states that correlate with many biological features as seen previously in other systems. Investigating patterns of states on a larger scale, we observe extended active and inactive domains corresponding with regions of low and high H3K27me3, and we characterize two types of border regions separating domains. Marked borders are associated with higher levels of H3K36me3 and germline active chromatin. These borders show spreading of H3K27me3 into neighboring active regions in animals with reduced H3K36me3 germline marking, suggesting that germline events contribute to domain definition. Regulatory borders are enriched for characteristics of transcriptional regulatory regions. The domain organization shows similarities to topological domains in humans and Drosophila, where insulator proteins such as CTCF show enrichment at boundaries. Using large modENCODE and ENCODE collections of transcription factor binding data, we find that the binding of most transcription factors is enriched at C. elegans borders and Drosophila and human topological boundaries, indicating that the previously observed topological boundary enrichment of insulator proteins is not specific to these factors. We propose that these regulatory regions may help to organize chromatin into domains.