Bucher EA et al. (1994) Seminars in Developmental Biology "Gastrulation in the nematode Caenorhabditis elegans."

Bucher EA, Seydoux GC

[Seminars in Developmental Biology, 1994]

Gastrulation in Caenorhabditis elegans has been described by following the movements of individual nuclei in living embryos by Nomarski microscopy. Gastrulation starts in the 26-cell stage when the two gut precursors, Ea and Ep, move into the blastocoele. The migration of Ea and Ep does not depend on interactions with specific neighboring cells and appears to rely on the earlier fate specification of the E lineage. In particular, the long cell cycle length of Ea and Ep appears important for gastrulation. Later in embryogenesis, the precursors to the germline, muscle and pharynx join the E descendants in the interior. As in other organisms, the movement of gastrulation permit novel cell contacts that are important for the specification of certain cell fates.

Bucher EA et al. (1991) Genetics "A genetic mosaic screen of essential zygotic genes in Caenorhabditis elegans."

Greenwald IS, Bucher EA

[Genetics, 1991]

We have devised a simple genetic mosaic screen, which circumvents the difficulties posed by phenotypic analysis of early lethal mutants, to analyze essential zygotic genes in Caenorhabditis elegans. The screen attempts to distinguish genes involved in cell type and/or lineage specific processes such as determination, differentiation or morphogenesis from genes involved in general processes such as intermediary metabolism by using the pattern of gene function to classify genes: genes required in one or a subset of early blastomeres may have specific functions, whereas genes required in all early blastomeres may have general functions. We found that 12 of 17 genes examined function in specific early blastomeres, suggesting that many zygotic genes contribute to specific early processes. We discuss the advantages and limitations of this screen, which is applicable to other regions of the C. elegans genome.

Bucher EA et al. (1991) International C. elegans Meeting "A GENETIC MOSAIC SCREEN OF ESSENTIAL ZYGOTIC GENES IN C. ELEGANS."

Greenwald IS, Bucher EA

[International C. elegans Meeting, 1991]

We have used a simple genetic mosaic screen to classify essential zygotic genes. This screen attempts to classify genes based upon whether the gene function iS required in all early blastomeres or only in specific early blastomeres (restricted lethal foci) for viability. We hypothesize that genes required in all early blastomeres may have more general functions, such as 'housekeeping', whereas genes required in specific early blastomeres may have specific functions, such as in determination, differentiation or morphogenesis. In contrast to other approaches, mosaic analysis does not rely upon lethal phenotypes and therefore may enable us to recognize mutations in genes with specific developmental functions even if they have variable or complicated lethal phenotypes. We generated 26 independent lethal mutations in the strain ncl-l (el 865) unc-36(e251) glp-l (q46); qDp3. The visible markers unc-36 and glp-l each have a specific focus of activity, in AB and P4, respectively. Rare somatic losses of qDp3 during early founder cell divisions can be recognized by a simple screen for viable Unc-36 or Glp-l animals. We analyzed 20 lethal mutations by the mosaic strategy. In contrast to the control strain, which gave all possible classes of mosaics, strains containing one of 7 lethal mutations never generated viable mosaics. We conclude that these mutations define genes that are required in all early blastomeres. In contrast, strains containing one of 13 mutations gave rise to specific patterns of mosaics suggesting that the gene activity is required in only a subset of early blastomeres for viability (restricted lethal foci). In addition, two strains that generated viable Unc-36 mosaics exhibited additional phenotypes in cells lacking expression of the gene, suggesting that an essential function has been separated from a nonessential function. Genes that have restricted foci are of interest to us since these may have specific roles in development; therefore we have begun further analysis of four genes of particular interest. Future analysis of individual genes will be important in understanding the types of genes identified in the genetic mosaic screen, and for evaluating the utility of the approach for future studies in this and other regions of the C. elegans genome. Furthermore, the functions of zygotic genes in early development is currently unclear. The high number of mutations (13/20) having specific lethal foci in our genetic mosaic screen suggests that many essential zygotic genes have specific developmental functions, if our original hypothesis is true. This inference is in contrast to the interpretation that most zygotic genes have housekeeping functions based upon the time of zygotic gene function and/or expression. Detailed genetic and molecular analysis is needed to gain an understanding of essential zygotic gene functions.

Bucher EA et al. (1990) Worm Breeder's Gazette "GENETIC ANALYSIS OF ZYGOTICALLY ACTING GENES ESSENTIAL FOR EARLY DEVELOPMENT IN C. ELEGANS"

Bucher EA, Greenwald IS

[Worm Breeder's Gazette, 1990]

We are using a genetic mosaic screen for mutations in zygotically acting genes that are essential for early development. This strategy allows us to distinguish gene products whose functions are required in specific lineages from gene products required for general cell viability (housekeeping functions). In contrast to other approaches, mosaic analysis does not rely upon lethal phenotypes, but upon focus of gene activity, and therefore may allow us to identify mutations in genes whose lethal phenotypes we may not anticipate. We hypothesized that this approach is reasonable since we can make the following predictions about the properties that different classes of essential, zygotically active genes may exhibit: Class I. genes whose functions are required in all lineages (housekeeping); Class II. genes whose functions are required everywhere but expression in part of the lineage is sufficient (diffusible housekeeping); Class III. genes whose functions are involved in determination and differentiation and required only in specific lineages; Class IV. genes having essential functions in certain lineages and nonessential functions in others. We have generated 31 independent lethal mutations in the strain ncl- 1 glp-1; d by Judith Austin). The EMS induced lethal mutations are linked to these recessive markers and complemented by the free duplication qDp3. The visible markers unc-36 and glp-1 have specific foci of activity in ABp and P4, respectively, allowing a rapid visual screen for rare somatic losses of qDp3 during early cell divisions. Losses of qDp3 are determined precisely using the cell autonomous nucleolar marker ncl-1 ( E. Hedgecock) visualized under Nomarski optics. The pattern of rare viable mosaics should reflect the requirement for gene activity. Mosaic analysis among a set of the 31 lethal strains that we have generated has distinguished mutations consistent with the predicted classes: some lethal strains never generate mosaics (Class I); a few strains generate viable mosaics in a restricted part of the lineage ( Class III); and two strains generate viable mosaics in a restricted part of the lineage and show an additional specific phenotype (Class IV); (see below). Although we have not yet seen any mosaic patterns consistent with Class II gene products, Bob Herman (personal communication) and Wightman and Ruvkun (WBG (1989) 11: 48) have observed mosaic patterns that we consider to be of this class. Strain GS192 appears to contain a mutation in a Class IV gene that has an essential function in the P1 lineage and a nonessential function in the AB lineage. Rare viable mosaics (picked as Uncs or semi Uncs) in GS192 result from losses in AB, ABp, ABpl, and ABpr . In contrast, we have never isolated a viable mosaic resulting from loss in the P1 lineage, suggesting that the expression of this gene product is absolutely required in these cells. However, a nonessential function was revealed by the abnormal morphology of neurons derived from AB in mosaic animals lacking qDp3 in these AB derived cells. Neurons derived from AB do not exhibit their characteristic granular appearance and do not have visible Nc1 nucleoli. GS161 also appears to encode a Class IV product. Unc and semi-Unc mosaic animals result from losses in either ABp, ABpl, or ABpr. This pattern suggests that expression of the gene product is absolutely required in the P1 lineage as well as in the ABa lineage. A nonessential function was revealed by abnormal vulval formation in mosaic animals. This observation is especially interesting since cells giving rise to the vulva descend from ABp. All mosaics are Egl-; some mosaics have no vulva, whereas other mosaics have an abnormal vulva. Preliminary mapping data place this allele left of ncl-1 where, to our knowledge, no genes affecting vulval development have been previously identified. The variable vulval phenotype may indicate either that this is not a null allele or, alternatively, may reflect some aspect of this gene product's requirement in the vulval lineages. These data argue that this genetic mosaic screen may indeed allow us to identify lethal mutations in zygotically expressed genes that are required for basic developmental processes. Furthermore, mosaic analysis not only allows us to distinguish among genes products having different foci of activity, but may also allow us to identify mutations in genes which may be too pleiotropic to recognize by criteria such as terminal phenotype. Some gene products of interest to us may have multiple, but specific roles in development (Cline, T.W. (1989) Cell 59: 231-234.) and thus may be classified as pleiotropic by classical criteria but may be identified in this genetic mosaic strategy as interesting. (Note: we have detected recombination between qDp3 and the chromosomes with some growth advantage for recombinants.)

Ramirez-Fajardo, Emilio et al. (2021) International Worm Meeting "Innate immune responses of Caenorhabditis elegans to the emerging pathogen Elizabethkingia anophelis"

Schmidt, Kristopher, Albayati, Wesam, List, Skylar, Ramirez-Fajardo, Emilio, Grossen, Sarah, Siderhurst, Matthew

[International Worm Meeting, 2021]

Elizabethkingia anophelis (Ea) is an emerging pathogen that causes sepsis, meningitis, and death in people with comorbidities. While usually innocuous, a recent community outbreak resulted in at least 20 deaths and 66 laboratory confirmed cases. The outbreak stemmed from the rapid formation of a novel and more virulent Ea sublineage created by a conjugative element that disrupted the muty DNA repair gene. The virulence associated factors, pathogenic mechanisms, and ecology of Ea are not known. C. elegans is a well-established model for the characterization of pathogen-host interactions. When we exposed mixed stage wild-type C. elegans to Ea (Ag1 strain), the animals exhibited decreased movement, slowed development, early larval death, and an overall decreased fecundity compared to animals exposed to E. coli OP50 (Ec). We also made the striking observation that worms exposed to Ea actively avoided Ea lawns. To understand the aversive response, we conducted two-choice assays between Ea and Ec in experiments using L1 and L4 animals. L1s not previously exposed to a food source initially chose Ea over Ec (-0.75 CI, Choice Index [CI] of -1.0 represents Ea selection) but actively selected Ec over time (+0.41 CI at 96hrs). L4 animals first reared on Ec chose Ec over Ea in two-choice assays (+0.49 CI). Importantly, we found that staged adults placed on Ea bacteria without another bacterial food source adopted a lawn avoidance phenotype, suggesting that they are repulsed from Ea even in the absence of Ec. These avoidance responses depend on an intact odorant detection system because lim-4(ky403) and tax-2(p671); tax-4(p678) mutant animals failed to avoid Ea lawns. We used solid-phase microextraction (SPME) gas chromatography mass spectrometry (GC-MS) to identify volatile odorant differences between Ea and Ec and observed a marked increase in the amount of indole in Ec compared to Ea. Indole is an attractive compound recently shown to modulate predator-prey interactions between C. elegans and bacteria. To examine the pathogenic mechanisms of Ea in C. elegans, we exposed N2, sek-1 (km4), tol-1(nr2033), dbl-1(nk3) and daf-2(e1370) animals to Ea and Ec. Wild-type animals displayed several pathological changes when grown on Ea and died faster than their Ec exposed counterparts. Decreased lifespan and increased pathologies were found in sek-1 while daf-2 animals appeared immune to Ea. The tol-1 and dbl-1 alleles did not influence Ea pathogenicity. To complement these immunity assays we also examined the antimicrobial activity of dod-24, irg-1 and T24B8.5 in C. elegans exposed to Ea using transcriptional reporter GFP strains. We observed decreases in the expression of T24B8.5 and increased but more variable changes in irg-1. Taken together these experiments suggest that Ea is pathogenic to C. elegans, opening new opportunities to investigate pathogen and host factors influencing Ea related disease.

Theresa M. Grana et al. (2007) International Worm Meeting "Cell-cell adhesion molecules regulate gastrulation in C. elegans."

Theresa M. Grana, Elisabeth A. Cox, Jeff Hardin

[International Worm Meeting, 2007]

Morphogenesis requires dynamic regulation of cell adhesion and the cytoskeleton. The first major morphogenetic movement in development is gastrulation. C. elegans gastrulation begins at the 26-cell stage with the migration of the two endodermal precursors Ea/Ep from the surface of the embryo into the interior. Ea/Ep migration provides a relatively simple system to examine the intersection of cell adhesion, cell signaling, and cell movement. Ea/Ep ingression depends on their specification and polarization, apical myosin accumulation and Wnt activated actin-myosin contraction that drives apical constriction and ingression (Nance et al., Wormbook, 2005; Lee et al., Current Biology, 2006). Here, we show that Ea/Ep ingression also requires cell-cell adhesion mediated by either HMR-1/cadherin or SAX-7/L1 CAM (L1 cell adhesion molecule). Either adhesion system is sufficient for Ea/Ep ingression, but loss of both together leads to a failure of apical constriction and ingression. In sax-7(eq1); hmr-1(RNAi) embryos we observe correct specification of Ea/Ep, based on timing of cell divisions and the presence of birefringent gut granules, and correct apical polarization of Ea/Ep, based on basolateral localization of PAR-2::GFP. Significantly, in sax-7(eq1); hmr-1(RNAi) embryos Ea/Ep fail to accumulate myosin (NMY-2::GFP) at their apical surfaces, but in either sax-7(eq1) or hmr-1(RNAi) alone, apical myosin accumulation is equivalent to wildtype. (In gastrulation stage embryos, HMR-1 (as indicated by HMP-2 staining) and SAX-7 are localized at all sites of cell-cell contact and do not appear on exposed cell surfaces.) SAX-7 and HMR-1 may function to localize myosin via signaling or by directing localization of myosin-binding molecules.

Grana TM et al. (2010) Dev Biol "SAX-7/L1CAM and HMR-1/cadherin function redundantly in blastomere compaction and non-muscle myosin accumulation during Caenorhabditis elegans gastrulation."

Cox EA, Hardin J, Grana TM, Lynch AM

[Dev Biol, 2010]

Gastrulation is the first major morphogenetic movement in development and requires dynamic regulation of cell adhesion and the cytoskeleton. Caenorhabditis elegans gastrulation begins with the migration of the two endodermal precursors, Ea and Ep, from the surface of the embryo into the interior. Ea/Ep migration provides a relatively simple system to examine the intersection of cell adhesion, cell signaling, and cell movement. Ea/Ep ingression depends on correct cell fate specification and polarization, apical myosin accumulation, and Wnt activated actomyosin contraction that drives apical constriction and ingression (Lee et al., 2006; Nance et al., 2005). Here, we show that Ea/Ep ingression also requires the function of either HMR-1/cadherin or SAX-7/L1CAM. Both cadherin complex components and L1CAM are localized at all sites of cell-cell contact during gastrulation. Either system is sufficient for Ea/Ep ingression, but loss of both together leads to a failure of apical constriction and ingression. Similar results are seen with isolated blastomeres. Ea/Ep are properly specified and appear to display correct apical-basal polarity in sax-7(eq1);hmr-1(RNAi) embryos. Significantly, in sax-7(eq1);hmr-1(RNAi) embryos, Ea and Ep fail to accumulate myosin (NMY-2Colon, two colonsGFP) at their apical surfaces, but in either sax-7(eq1) or hmr-1(RNAi) embryos, apical myosin accumulation is comparable to wild type. Thus, the cadherin and L1CAM adhesion systems are redundantly required for localized myosin accumulation and hence for actomyosin contractility during gastrulation. We also show that sax-7 and hmr-1 function are redundantly required for Wnt-dependent spindle polarization during division of the ABar blastomere, indicating that these cell surface proteins redundantly regulate multiple developmental events in early embryos.

Zhang W et al. (2020) J Food Sci "Effects of ethyl acetate fractional extract from Portulaca oleracea L. (PO-EA) on lifespan and healthspan in Caenorhabditis elegans."

Zhang W, Wang H, Liu RH, Zheng B, Li T, Deng N

[J Food Sci, 2020]

Portulaca oleracea L. (PO), with abundant natural bioactive phytochemicals, exhibits potential bioactivities and pharmacological activities. However, the mechanisms of action of PO on anti-aging effect remain unclear. In this study, the ethyl acetate fractional extract from PO (PO-EA) was obtained by fractionation of solvent extractions, and its effect on lifespan was assessed using the Caenorhabditis elegans (C. elegans). Results showed that PO-EA could significantly increase the lifespan of C. elegans by 5.31, 12.67, and 16.47% at the doses of 250, 500, and 1,000g/mL, respectively. Moreover, PO-EA significantly promoted the mobility of C. elegans without obvious side effects such as changing body length or decreasing fecundity of the nematodes. Further study demonstrated that PO-EA could enhance the stress resistance in C. elegans via improving the activities of superoxide dismutase and catalase, and diminishing the contents of reactive oxygen species and malondialdehyde. The gene expression of daf-12, daf-16, sod-3, skn-1, cat-1, mev-1, akt-1, and sek-1 were upregulated in C. elegans after administrated by PO-EA. This study indicated that PO-EA plays a vital role in extending lifespan and healthspan in C. elegans, and the underlying mechanism of action might be attributed to Insulin/IGF-1-like signaling pathways. Therefore, PO-EA could be served as a potential candidate for anti-aging functional food. PRACTICAL APPLICATION: Portulaca oleracea L. (PO) is an edible vegetable that could be used as functional food to exert health benefits for humans such as neuroprotective, antioxidant, anticancer, and anti-aging effects. Therefore, our findings would provide a strategy to promote the comprehensive utilization of ethyl acetate extract from PO with additional health benefits.

T Elbl et al. (1999) International C. elegans Meeting "mup-4, a Gene Required for Epidermal Morphogenesis and Muscle Position, is a Member of a Novel Family of Transmembrane Proteins."

J Ward, A Franzini-Armstrong, EA Bucher, T Elbl, L Hong

[International C. elegans Meeting, 1999]

mup-4 is a member of a new family of genes hypothesized to mediate cell-cell and/or cell-matrix interactions gene in C. elegans. mup-4 and mua-3 have recently been cloned and are homologues. Mutants for these genes show related, but distinct phenotypes: mup-4 mutants exhibit defects in muscle position at 3-fold 1 , whereas mua-3 mutants display muscle position defects during larval stages 2 . In addition, mup-4 mutants have epidermal defects in embryonic enclosure and vulval morphogenesis (in genetic mosaics). mup-4 and mua-3 2 encode large transmembrane proteins having novel, largely unrelated, intracellular domains, and similar extracellular domains with EGF-like, type XII collagen, and novel, shared, motifs. The phenotypic and molecular data are consistent with functions in cell-extracellular matrix (ECM) or cell-cell adhesion and/or signaling pathways. We are currently testing to distinguish among possible models for MUP-4 function by: (i) determining the temporal, spatial and subcellular localization of MUP-4; (ii) examining the ultrastructural defects in mup-4 mutants; and (iii) testing for interacting partners with yeast 2-hybrid and genetic interaction studies. Thus far, our ultrastructural studies demonstrate that muscle detachment is associated with a lesion between the hypodermis and the exoskeletal cuticle, and our expression studies demonstrate that mup-4 is expressed in the hypodermis overlying muscles. Furthermore, consistent with the proposed role in morphogenesis, we find that mup-4 is also expressed in embryonic hypodermal cells, prior to embryo enclosure, and in the anchor cell, a cell involved in vulval morphogenesis. One possible hypothesis to explain these phenotypic and expression data is that MUP-4 functions: i) in hypodermal adhesion or polarity and thus influences epidermal morphogenetic events and ii) by adhering the apical membrane of the hypodermis to the cuticular ECM and transmitting the force of muscle contraction to the exoskeleton. 1 Gatewood, B. K., and E. A. Bucher. 1997. Genetics 146:165-83. 2 J. Plenefisch and E. Hedgecock, pers. com.

Saul N et al. (2011) J Nat Prod "Diversity of polyphenol action in Caenorhabditis elegans: between toxicity and longevity."

Pietsch K, Sturzenbaum SR, Menzel R, Steinberg CE, Saul N

[J Nat Prod, 2011]

The model organism Caenorhabditis elegans was utilized to determine, in vivo, the mode(s) of action of four plant polyphenols, namely, tannic acid (TA), gallic acid (GA), ellagic acid (EA), and catechin (CT). The determination of lifespan, stress resistance, growth, reproduction, eating-related behaviors, antioxidative capacities, and lifespan assays with the mev-1 and the eat-2 mutants as well as in the presence of dead bacteria provided new insights into their action. All four compounds prolonged lifespan, but only TA and CT mediated distinct stress protection. Longevity is unlikely the result of antioxidant capacities but rather due to calorie restriction imitating and hormetic properties in the case of TA and EA or antimicrobial capacities of GA and EA. Furthermore, the prominent "disposable soma theory" is only partly reflected by these polyphenols. In summary, this study underlines the diversity of polyphenolic phytochemicals and their mechanistic background.