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[
International Worm Meeting,
2021]
Recording neural activity at single cell resolution during unrestrained behavior holds tremendous potential for investigating the C. elegans neural code on a global scale. As fluorescent calcium indicators and 3D microscopy speeds have improved, recording from 100's of cells in moving worms has become feasible. However, 2 problems remain 1) being able to extract robust information from individual cells in a moving worm, and 2) knowing the defined identity of each individual tracked cell. Recently, a novel C. elegans strain termed NeuroPAL was developed that labels individual neuronal identities via a stereotypical multi-color fluorescence map. To harness the power of this worm we developed a high-speed multispectral volumetric microscopy platform with sub-cellular resolution, optimized for the NeuroPAL worms. Our SCAPE microscopy-based approach uses a scanning oblique light sheet which provides low phototoxicity and optical sectioning capabilities in a convenient single-objective geometry compatible with common C. elegans sample mounting procedures. The system's multi-laser launch, spectral image splitter and high-speed intensified camera make it possible to rapidly acquire a fully 3D NeuroPAL image in under 0.3 s. These scans can be interspersed with dual channel imaging of GCaMP and RFP with 0.33 x 0.67 x 0.25 microm sampling density over a 310 x 210 microm FEP-covered agarose arena at 13 volumes per second. The resulting data suggests much simpler tracking of uniquely identifiable cells throughout the worm, and analysis of the cellular calcium dynamics during free behavior.
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[
International Worm Meeting,
2015]
Parafilm M® is a thin thermoplastic used to seal a variety of containers in scientific laboratories. It is commonly used to seal Nematode Growth Media (NGM) culture plates to prevent microbial contamination and media dehydration. However, the effects on C. elegans of wrapping culture plates with Parafilm M® during experiments are unknown. Parafilm M® may limit gas exchange between the external and culture environment, potentially affecting the biology and life history of C. elegans, including its larval growth rate, viability, fecundity, lifespan, and behavior. In particular, wrapping culture plates with Parafilm M® may produce a hypoxic (low oxygen) environment compared to plates with no Parafilm M® (normoxic). Anoxic (no oxygen) and hypoxic conditions have been shown to change the metabolism, development, and longevity of C. elegans.Our research aims to determine the effects of wrapping NGM culture plates with Parafilm M® on C. elegans. We hypothesized that worms cultured on plates wrapped in Parafilm M® would exhibit a slower rate of larval growth and increased mortality compared to worms grown in normoxic conditions. Synchronized L1 worms were individually transferred to culture plates and incubated within an anoxic environment, hypoxic environment, normoxic environment, or wrapped one time with Parafilm M®. BD GasPaks™ were used to create anoxic and hypoxic environments, and normoxic culture conditions consisted of unsealed plates. Larval growth rate and mortality were measured 5 times over 48 hours. We found no significant difference in the growth rate between worms cultured in normoxic conditions and on plates wrapped with Parafilm M®. However, the growth rate between worms cultured on plates wrapped with Parafilm M® and worms in anoxic and hypoxic conditions was significantly higher. Mortality was significantly higher in worms cultured in anoxic conditions, but was not significantly different among the other three environmental conditions. Our data suggest that wrapping C. elegans culture plates one time with Parafilm M® does not affect the larval growth rate or viability. Future studies will focus on additional biological and life history metrics, such as fecundity and lifespan, to verify that wrapping with Parafilm M® has no unexpected effects on the outcomes of C. elegans studies.
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[
International Worm Meeting,
2007]
Heme serves as a cofactor for a number of proteins involved in key metabolic processes. In eukaryotes, heme synthesis occurs in the mitochondria by an evolutionarily conserved multi-step pathway. Hemes are hydrophobic and thus insoluble in the aqueous environment of the cell. Moreover, free heme is cytotoxic because of peroxidase activity. We therefore hypothesize that intracellular pathways exist for trafficking of heme from the site of synthesis in the mitochondria to various cellular destinations. However, identification of these heme transport pathways has been difficult because heme synthesis is regulated by multiple effectors and is tightly coordinated with apo-protein synthesis. We have previously shown that C. elegans and related helminths do not make heme albeit requiring exogenous heme for normal metabolic processes. Importantly, C. elegans show a biphasic response for heme; worms are growth-arrested at 1.5 <font face=symbol>m</font>M and at 800 <font face=symbol>m</font>M heme. These results suggest that although worms are obligate heme auxotrophs they are likely to have all the pathways for heme utilization beyond the point of heme synthesis. To identify pathways for heme transport in C. elegans, we exploited their biphasic response to heme by screening for mutants that could survive heme toxicity. We screened 300,000 haploid genomes and isolated 13 mutants at 800 <font face=symbol>m</font>M heme in liquid axenic medium. Based on the mutants growth profile in medium containing low and high heme, we categorized the mutants into three broad phenoclusters: class A, class B and class C. Class A mutants grew robustly under low and high (800 and 1000<font face=symbol>m</font>M) heme, Class B mutants grew exceptionally well under low heme, moderately well at 800 <font face=symbol>m</font>M heme, and not at all at 1000 <font face=symbol>m</font>M heme, and Class C mutants grow moderately well under high heme (800<font face=symbol>m</font>M), but exhibit normal growth under low heme. The mutants were further sub-clustered by using gallium protoporphyrin (GaPP), a toxic heme analog. Complementation analyses revealed that these 13 mutants fall into five complementation groups. Genetic mapping by recombination localized the mutants from each complementation group to chromosome III. We are now producing a high resolution map to define a genetic interval and pinpoint the exact nature of the molecular lesion in these mutants.
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[
European Worm Meeting,
2002]
M. nematophilum is a novel pathogen of C. elegans recently described by J. Hodgkin et al. (1). The bacterium is able to attach to the post-anal region of C. elegans and to induce massive swelling of the underlying tissues by an unknown mechanism. The disease causes constipation and slows growth of affected worms. M. nematophilum belongs to the Gram-positive coryneform group of bacteria and is poorly characterised.
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[
Development & Evolution Meeting,
2008]
The C. elegans postembryonic mesodermal lineage, the M lineage, is a powerful model system to study mesodermal patterning and cell fate specification at single cell resolution. The M lineage arises from a single pluripotent cell, the M mesoblast, during embryogenesis. In hermaphrodites, the M cell undergoes a series of postembryonic cell divisions to produce 18 cells: 14 body wall muscles (BWMs), 2 coelomocytes (CCs), and 2 sex myoblasts (SMs). We and others have previously identified a handful of transcription factors important for the proper development of this lineage. In order to identify additional transcription factors that play a role in the M lineage, we have generated a feeding RNAi library that targets a majority of the predicted transcription factors encoded in the C. elegans genome and conducted an RNAi screen using cell type-specific GFP reporters in the M lineage. From this screen, we identified a novel set of 32 transcription factors that, upon RNAi knockdown, give reproducible phenotypes in the M lineage. Among these 32 transcription factors, four are important for patterning and fate specification of the early M lineage, while the rest appear to play a role in fate decisions in the SM lineage. We have primarily focused on
let-381, which encodes a forkhead transcription factor that is essential for C. elegans development.
let-381(RNAi) causes a dorsal to ventral fate transformation in the M lineage. We have found that a
let-381::gfp translational fusion is expressed in the dorsal M lineage. Previous studies from our lab have shown that SMA-9, the Sma/Mab TGF-beta and LIN-12/Notch signaling pathways are involved in dorsal/ventral patterning of the M lineage. We are currently investigating the relationship between
let-381 and these pathways.
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[
International Worm Meeting,
2007]
The C. elegans post-embryonic mesodermal lineage arises from a single precursor cell, the M mesoblast, which will diversify to generate distinct dorsal and ventral cell types. The dorsal daughter of M gives rise to a subset of body wall muscles and two non-muscle coelomocytes, whereas the ventral daughter of M gives rise to two sex myoblasts in addition to a subset of body wall muscles. Mutations in the C. elegans Schnurri homolog
sma-9 cause ventralization of the M lineage. We have previously shown that SMA-9 antagonizes the Sma/Mab TGF-beta signaling pathway to promote dorsal M lineage fates (Foehr et al., 2006). Interestingly, loss-of-function mutations in the Notch receptor homolog
lin-12 cause dorsalization of the M lineage (Greenwald et al., 1983), an exact opposite phenotype of
sma-9 mutants. We have found that while LIN-12 protein is present in both the dorsal and ventral M lineage cells, the ligands for LIN-12, LAG-2 and APX-1, are asymmetrically localized in cells adjacent to ventral M-derived cells, and they function redundantly in promoting ventral M lineage fates. To investigate how LIN-12/Notch signaling interacts with SMA-9 and the Sma/Mab TGF-beta pathway in regulating M lineage patterning, we generated double and triple mutant combinations among
lin-12,
sma-9 and
dbl-1 (the ligand for the Sma/Mab TGF-beta pathway) and examined their M lineage phenotypes. Our results suggest that the LIN-12/Notch pathway and the Sma/Mab TGF-beta pathway function independently in regulating dorsoventral patterning of the M lineage, with LIN-12/Notch required for ventral M lineage fates, and SMA-9 antagonism of TGF-beta signaling required for dorsal M lineage fates. Our work provides a model for how combined Notch and TGF-beta signaling regulates the developmental potential of two equipotent cells along the dorsoventral axis.
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[
International Worm Meeting,
2005]
Microbacterium nematophilum (M. nem) is a Gram-positive bacterium belonging to the coryneform group, which was discovered as a result of its ability to cause disease and morphological alteration in C. elegans (Hodgkin et al., 2000). Nematode cultures have been accidentally contaminated by M. nem and consequently become diseased on several independent occasions. The pathogenic bacteria colonize the rectum of susceptible worms and cause localized swelling, constipation and growth impairment, but they do not usually prevent survival and reproduction of infected worms. Similar pathology is seen after infection of C. briggsae and C. remanei. Recent taxonomic and genomic analyses (Kiontke et al., 2004) have defined new candidate species and a robust phylogeny for the Caenorhabditis genus. Ten species that can be easily grown on standard NGM agar/E. coli plates are now available (several were kindly provided to us by Karin Kiontke and David Fitch), and all have been tested for susceptibility to M. nem. In contrast to the effects on the four species of the 'elegans' group (C. elegans, C. briggsae, C. remanei, C. sp CB5161), M. nem is lethal to the other six species. Larvae exposed to either a mixed E. coli/M. nem lawn, or a pure M. nem lawn, are unable to mature, and usually experience paralysis, lysis and death after one or two days. For some but not all of the species, the dying larvae exhibit a Dar (swollen tail) phenotype. These observations suggest that M. nem is potentially a serious pathogen for rhabditid soil nematodes, and that members of the elegans group have evolved defenses that ameliorate the infection. Analysis of the C. elegans response has identified components of these defenses (see related abstracts). In most cases where more than one independent isolate of a species was tested, similar results were obtained. However, isolates of C. briggsae are noticeably heterogeneous, ranging from almost complete resistance to extreme sensitivity. Avirulent derivatives of the M. nem type strain, CBX102, have been generated, which are unable to cause disease in C. elegans. Some of these avirulent mutants have been tested on the more sensitive species, and found to be similarly non-virulent, which suggests that related mechanisms are involved in causing disease (in C. elegans) and death (in the more sensitive species).
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[
Development & Evolution Meeting,
2006]
mls-2 encodes a HMX homeodomain protein that plays critical roles in the C. elegans postembryonic mesodermal lineage, the M lineage.
mls-2 is expressed in the M lineage as well as a few other cell types, such as several pairs of head neurons including ASK and AIM. To uncover mechanisms involved in regulating the expression of
mls-2, we generated a series of promoter deletions and identified an M lineage enhancer (position -2221 to -2076) that is required for
mls-2 expression in the M lineage. We further identified several essential elements within this M enhancer. Among these elements are two putative Exd/Abd-B binding sites that are conserved in C. briggsae. We have found that
mls-2 expression in the M lineage is dependent on the C.elegans Exd/Pbx homolog CEH-20. We are currently testing whether CEH-20 directly binds to the
mls-2 promoter and whether CEH-20 regulates
mls-2 expression through acting together with the Abd-B class Hox proteins, EGL-5/PHP-3/NOB-1, and the Hth/Meis homolog, UNC-62.
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[
International Worm Meeting,
2017]
Extracellular vesicles are emerging as an important aspect of intercellular communication by delivering a parcel of proteins, lipids even nucleic acids to specific target cells over short or long distances (Maas 2017). A subset of C. elegans ciliated neurons release EVs to the environment and elicit changes in male behaviors in a cargo-dependent manner (Wang 2014, Silva 2017). Our studies raise many questions regarding these social communicating EV devices. Why is the cilium the donor site? What mechanisms control ciliary EV biogenesis? How are bioactive functions encoded within EVs? EV detection is a challenge and obstacle because of their small size (100nm). However, we possess the first and only system to visualize and monitor GFP-tagged EVs in living animals in real time. We are using several approaches to define the properties of an EV-releasing neuron (EVN) and to decipher the biology of ciliary-released EVs. To identify mechanisms regulating biogenesis, release, and function of ciliary EVs we took an unbiased transcriptome approach by isolating EVNs from adult worms and performing RNA-seq. We identified 335 significantly upregulated genes, of which 61 were validated by GFP reporters as expressed in EVNs (Wang 2015). By characterizing components of this EVN parts list, we discovered new components and pathways controlling EV biogenesis, EV shedding and retention in the cephalic lumen, and EV environmental release. We also identified cell-specific regulators of EVN ciliogenesis and are currently exploring mechanisms regulating EV cargo sorting. Our genetically tractable model can make inroads where other systems have not, and advance frontiers of EV knowledge where little is known. Maas, S. L. N., Breakefield, X. O., & Weaver, A. M. (2017). Trends in Cell Biology. Silva, M., Morsci, N., Nguyen, K. C. Q., Rizvi, A., Rongo, C., Hall, D. H., & Barr, M. M. (2017). Current Biology. Wang, J., Kaletsky, R., Silva, M., Williams, A., Haas, L. A., Androwski, R. J., Landis JN, Patrick C, Rashid A, Santiago-Martinez D, Gravato-Nobre M, Hodgkin J, Hall DH, Murphy CT, Barr, M. M. (2015).Current Biology. Wang, J., Silva, M., Haas, L. A., Morsci, N. S., Nguyen, K. C. Q., Hall, D. H., & Barr, M. M. (2014). Current Biology.
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[
International C. elegans Meeting,
1999]
Bodywall muscles are derived from four of the embryonic founder cells as well as the postembryonic M blast cell. We are studying patterning of both the embryonic and postembryonic muscles in order to understand how myogenic cell fates are specified during development. As an entry point to study muscle patterning during embryonic development, we characterized enhancer elements from the
hlh-1 regulatory region that are able to drive reporter expression in muscle precursors of the MS, D and C lineages. Analyses of these elements suggested regulatory roles of the Hox genes and the
hlh-1 gene itself. These analyses also suggested regulatory mechanisms involving unknown bZIP, bHLH and novel transcription factors. We are currently in the process of genetically identifying these factors. The postembryonic M lineage provides an alternative model to study myogenic fate specification. The M lineage gives rise to 14 bodywall muscles, 2 coelomocytes and 16 sex muscles. A number of genes had previously been identified to function in patterning the M lineage, including the Hox gene
mab-5 and the bHLH transcription factor twist (1, 2, 3). The role of the Hox factors in patterning the M lineage has been something of a mystery:
mab-5 is expressed during the entire M lineage, but
mab-5 mutants cause only limited lineage transformation. We will describe a series of experiments indicating a more central role for the Hox genes in activating twist and specifying the M lineage. We found that
lin-39 mab-5 double mutants fail to activate twist and completely lack products of the M lineage. Expression (either ectopic or in a normal pattern) of either Hox gene is sufficient to activate twist expression. However, twist activation is not sufficient for specification of the M lineage. Current efforts are directed towards identification of other factors involved in specifying the M lineage. 1. Kenyon, C. (1986), Cell 46: 477-487 2. Harfe B. D., Vaz Gomes A., Kenyon C., Liu J., Krause M., Fire A. (1998) Genes & Dev. 12: 2623-2635 3. A. Corsi, S. Kostas, E. Jorgensen, A. Fire, and M. Krause (poster)