Maine EM et al. (1994) Worm Breeder's Gazette "Mutations That Enhance glp-1 Identify Genes Required For Various Aspects of Germline Development."

Gelber MB, Smardon AM, Shu P, Qiao L, Maine EM, Lissemore JL

[Worm Breeder's Gazette, 1994]

Mutations that enhance glp-1 identify genes required for various aspects of germline development. Eleanor Maine, Li Qiao, Jim Lissemore-, Pei Shu, Anne Smardon, and Melanie Gelber. Biology Dept., Syracuse University, Syracuse, NY 13244 and Biology Dept., John Carroll University, Cleveland, OH 44118.

Erik Andersen et al. (2006) Development & Evolution Meeting "lst-3 Negatively Regulates the Vulval Cell-fate Decision"

Bob Horvitz, Melanie Worley, Erik Andersen, Scott Clark

[Development & Evolution Meeting, 2006]

The C. elegans vulva is formed by the descendants of three of six equipotent hypodermal blast cells. Two distinct vulval cell fates are specified by the actions of at least two conserved antagonistic signaling pathways, Ras and Notch. Mutations affecting these pathways cause the generation of ectopic vulval cells and a multivulva (Muv) phenotype. To understand more about factors controlling the vulval cell-fate decision, we performed a screen for suppressors of the synthetic multivulva (synMuv) phenotype. The synMuv genes are grouped into at least three functionally redundant classes, A, B and C, that negatively regulate the specification of vulval cell fates. Animals mutant for one or more genes within the same class are non-Muv, whereas animals mutant for genes within any two classes are Muv. We identified n2070 as a dominant suppressor of the synMuv phenotype of lin-15AB(n765) mutants. Our gene dosage experiments suggest that the dominant phenotype conferred by n2070 is caused by an increase in wild-type function. We mapped n2070 to a 120-gene interval on LGIV and then used RNAi to inactivate the genes in the interval to eliminate the synMuv suppression phenotype of n2070. RNAi of only lst-3 (lst, lateral signaling target) reverted the synMuv suppression phenotype caused by n2070. In a screen for cis-dominant suppressors of n2070, we identified three independent isolates with nonsense mutations in lst-3. n2070 causes a proline-to-leucine substitution in a putative DNA-binding domain of LST-3. The Greenwald laboratory identified lst-3 as a possible transcriptional target of LAG-1, a lin-12 Notch effector, and suggested that lst-3 mediates the interplay between the Ras and Notch pathways. lst-3 encodes a transcription factor similar to mammalian CARP-1, a cell cycle and apoptosis regulator. We isolated a deletion allele of lst-3. This allele confers a synMuv phenotype in combination with mutations in class A or C but not with mutations in class B synMuv genes. Through gene expression analysis of gain-of-function and loss-of-function lst-3 mutants, we have identified candidate LST-3 target genes. We will discuss how the modulation of lst-3 function might affect the Ras and Notch pathways.

JA Waddle et al. (1999) International C. elegans Meeting "Non-destructive, 3-D imaging of GFP-tagged proteins throughout C. elegans embryogenesis"

JA Waddle, RH Waterston

[International C. elegans Meeting, 1999]

A powerful feature of C. elegans research is the ability to determine mutant phenotypes at the resolution of single cells. The advent of green fluorescent protein (GFP) provides means to carry the single cell analysis one step further, that is, to characterize critical events that take place during a short interval within a cell cycle. Improvements in computers and imaging devices motivated us to develop a 3-dimensional image acquisition system capable of merging traditional embryonic cell lineage analysis with the latest GFP technology. To gauge the utility of our microscope setup, we imaged a transgenic line carrying a fusion between GFP and an isoform of histone H1 (kindly provided by Melanie Dunn and Geraldine Seydoux). The most important outcome of our efforts is the ability to non-destructively (e.g the embryos hatch and are fertile) collect a 3-dimensional time lapse fluorescence image sequence starting at first cleavage and spanning more than 8 hours of development. The histone H1::GFP images greatly facilitate the tracking of nuclear movements and cell divisions in the bottom half of later stage embryos. The application of computational methods, to reduce the effects of out of focus light, greatly improves image detail and the signal-to-noise ratio. Current efforts are focused on custom software capable of tracking the 3-dimensional distribution and behavior of the histone H1::GFP labeled nuclei, that is, automated embryonic cell lineage analysis. In summary, several technologies have coalesced to provide non-damaging means to track the 3-dimensional distribution of important molecules during embryogenesis. Such technology will complement existing methods for phenotypic analysis in an era when both the embryonic cell lineage and the genome descriptions are complete.

Siddiqui, Shahid S. et al. (2013) International Worm Meeting "Antimicrobial compound screens using C. elegans model system for periodontal pathogens -."

Siddiqui, Shahid S., Al-Beyari, Mohammad, Faskhani, Fathy A.

[International Worm Meeting, 2013]

Chronic periodontal disease (PD) is an inflammatory condition that is highly widespread. Periodontal disease is a result of sub-gingival plaque pathogens, leading to alveolar bone loss. PD is also associated with diabetes, obesity, cardiovascular disease, respiratory disease, rheumatic arthritis and pre-term birth. The mechanism of periodontal infection is poorly understood due to the complexity of microbial factors, and highly complex inflammatory and anti-inflammatory signaling between pathogens and the host. New microbial pathogens are emerging that are resistant to conventional antibiotics; thus there is a need for drugs that are new class of antibiotics, and novel compounds that reduce pathogenicity of invading microbes. Pioneering studies in Frederick Ausubel's Laboratory (Harvard Medical School) have shown the effective use of C. elegans as a simple and powerful genetic model to screen for antimicrobial compounds, such as the discovery of RPW-24 that protects C. elegans from bacterial infections by modulating the p38 MAPK, and ATF7 transcription regulatory pathways [Pukkila-Worley et al. 2012]. We have previously used C. elegans to screen chemicals by using the nematode egg laying assay, and have identified novel compounds that affect egg laying behavior in a high throughput screen of commercially available chemical library [Siddiqui et al., C. elegans meeting UCLA, 2011]. We are screening chemical libraries in C. elegans for novel compounds that may inhibit the growth of oral pathogens such as Pseudomonas aeruginosa, found in the periodontal infections; and look for modulators of NSY-1, SEK-1, PMK-1 Mitogen Activated Protein (MAP) Kinase pathway that is comparable to the mammalian ASK-1(MAP kinase kinase kinase)/MKK3/6 (MAP kinase kinase) p38 MAP kinase signaling pathway. To validate the discovery of novel compounds in C. elegans high throughput screens for periodontal pathogens, we will use in vitro PLC (periodontal ligament cell) cell culture screens. Results will be presented on the efficacy of screened compounds on oral pathogens specific to chronic periodontal disease.

Dunn MA et al. (1999) Worm Breeder's Gazette "A PIE-1-BASED VECTOR FOR MATERNAL EXPRESSION OF GFP FUSIONS."

Reese KJ, Seydoux GC, Dunn MA

[Worm Breeder's Gazette, 1999]

We have used sequences from the pie-1 gene to construct a vector designed to express GFP fusions in the adult germline and in early embryos. The pie-1 gene encodes a maternal RNA transcribed in the adult germline and inherited maternally in embryos (Mello et al., 1992, 1996; Tenenhaus et al., 1998). We have shown previously that an 8kb genomic fragment from the pie-1 locus is sufficient to tag PIE-1 with GFP and rescue a pie-1 mutation (Fig. 1; Dunn et al., Reese et al., 1998 East Coast Worm Meeting Abstracts). We now have modified this construct to allow expression of heterologous GFP fusions. The resulting vector (Fig. 2) contains the pie-1 promoter, the pie-1 3UTR, and the pie-1 large intron, all of which appear to be important for robust expression in the adult germline and in early embryos. We have tested this new vector in 4 configurations: as is (GFP alone), and with one of three different open-reading frames inserted at the SpeI site after the GFP: his-11 (histone H2B), C36E8.5 (beta tubulin), and act-1 (actin). These constructs were injected into N2 hermaphrodites using the Fire labs complex array injection method which permits germline expression of transgenes (Kelly et al., 1997, Genetics 146: 227-238). For all constructs, we obtained a number of lines (10% to 50% of all roller lines) with GFP expression in the germline and in early embryos. In most lines, expression was restricted to the F2 generation after injection. In all cases, the subcellular localization observed matched that expected for each fusion: throughout cells for GFP alone, on DNA for GFP:histone H2B, on centrosomes and meiotic (and more weakly mitotic) spindles for GFP:beta-tubulin, and on cell cortices and cell division remnants for GFP:actin. (In one GFP:actin line, we also saw nuclear GFP). In addition to germline expression, all lines also expressed GFP in a number of somatic cells. We are hopeful that this vector could be used by others to express additional GFP fusions in the germline and in early embryos. Anyone interested can obtain the pie-1 vector from Melanie Dunn at mdunn@jhmi.edu. Fig. 1. Fig. 2.

Waddle JA et al. (1998) East Coast Worm Meeting "USING GREEN CHROMOSOMES TO AUTOMATE EMBRYONIC CELL LINEAGE ANALYSIS"

Waterston RH, Waddle JA

[East Coast Worm Meeting, 1998]

While early embryonic cell lineage analysis is routine (1,2,3), tracking small nuclei in the bottom half of >100 cell embryos is not robust and requires considerable time and expertise (4). We hope to exploit green fluorescent protein technology (5) to develop methods for more robust, faster and highly automated lineage analysis. One potential method relies on two key components. First, Melanie Dunn and Geraldine Seydoux provided a histone H1::GFP transgene which permits observation of chromosomes in living embryos. Second, we developed a microscope image acquisition system capable of taking 64 fluorescent optical sections, 10 times per cell cycle, throughout embryogenesis, at light levels commensurate with normal development. Navigation of simultaneously collected histone::GFP fluorescence and Nomarksi DIC 4D data sets reveals that fluorescence lineage analysis is much easier than scoring divisions by Nomarksi optics. The discrimination of individual nuclei, especially in the bottom half of late embryos, is greatly improved by image processing to reduce the effects of out of focus light. We took advantage of the improved image quality to write a program that will identify the number and 3-dimensional position of each nucleus at a given time point. We are now developing software that compares the position of nuclei at subsequent time points in order to detect cell deaths, divisions and migrations. The output from the tracking program will be in two forms: a traditional lineage diagram showing the time and relative orientation of each embryonic cell division or death (until movement at 400 min.); and a series of 3-dimensional coordinates over time that can be used to show a color coded animation of the recording from any perspective. Finally, an additional technical barrier to automated cell lineage analysis is to identify a set of histone H1 genes that, in composite, labels chromosomes in every embryonic cell. The existing histone H1::GFP fusion (his-24 gene) is not highly expressed until the 28-cell stage, and is not expressed in a small subset of cells, including the germline precursors. We plan to fuse GFP to the remaining histone H1 genes to determine which are expressed in the HIS-24::GFP negative cells. The automated lineage analysis system should greatly facilitate phenotypic analysis of cell fate mutants and the subsequent dissemination of such information in a viewable, database format. 1. Hird and White, JCB 121:1343(1994). 2. Fire, CABIOS 10:443(1994). 3. Thomas et al., Science 273:603(1996). 4. Schnabel et al., Dev. Biol. 184:234(1997). 5. Chalfie et al., Science 263:802(1994).

Waddle JA et al. (1998) Midwest Worm Meeting "USING GREEN CHROMOSOMES TO AUTOMATE EMBRYONIC CELL LINEAGE ANALYSIS"

Waterston RH, Waddle JA

[Midwest Worm Meeting, 1998]

While early embryonic cell lineage analysis is routine (1,2,3), tracking small nuclei in the bottom half of >100 cell embryos is not robust and requires considerable time and expertise (4). We hope to exploit green fluorescent protein technology (5) to develop methods for more robust, faster and highly automated lineage analysis. One potential method relies on two key components. First, Melanie Dunn and Geraldine Seydoux provided a histone H1::GFP transgene which permits observation of chromosomes in living embryos. Second, we developed a microscope image acquisition system capable of taking 64 fluorescent optical sections, 10 times per cell cycle, throughout embryogenesis, at light levels commensurate with normal development. Navigation of simultaneously collected histone::GFP fluorescence and Nomarksi DIC 4D data sets reveals that fluorescence lineage analysis is much easier than scoring divisions by Nomarksi optics. The discrimination of individual nuclei, especially in the bottom half of late embryos, is greatly improved by image processing to reduce the effects of out of focus light. We took advantage of the improved image quality to write a program that identifies the number and 3-dimensional position of each nucleus at a given time point. We are now developing software that compares the position of nuclei at subsequent time points in order to detect cell deaths, divisions and migrations. The output from the tracking program will be in two forms: a traditional lineage diagram showing the time and orientation of each embryonic cell division (until movement at 400 min.); and a series of 3-dimensional coordinates over time. The nuclear coordinates can be used to show a color coded animation of embryonic development from any perspective. Finally, an additional technical barrier to automated cell lineage analysis is to identify a set of histone H1 genes that, in composite, labels chromosomes in every embryonic cell. The existing histone H1::GFP fusion (his-24 gene) is not highly expressed until the 28-cell stage, and is not expressed in a small subset of cells, including the germline precursors. We plan to fuse GFP to the remaining histone H1 genes to determine which are expressed in the HIS-24::GFP negative cells. The automated lineage analysis system should greatly facilitate phenotypic analysis of cell fate mutants and the subsequent dissemination of such information in a viewable, database format. 1. Hird and White, JCB 121:1343(1994). 2. Fire, CABIOS 10:443(1994). 3. Thomas et al., Science 273:603(1996). 4. Schnabel et al., Dev. Biol. 184:234(1997). 5. Chalfie et al., Science 263:802(1994).