scholarly journals Combined flow cytometry and high throughput image analysis for the study of essential genes in Caenorhabditis elegans

2017 ◽  
Author(s):  
Blanca Hernando-Rodríguez ◽  
Annmary Paul Erinjeri ◽  
María Jesús Rodríguez-Palero ◽  
Val Millar ◽  
Sara González-Hernández ◽  
...  
Keyword(s):  
Image Analysis ◽  
Caenorhabditis Elegans ◽  
High Throughput ◽  
Rnai Screen ◽  
Essential Genes ◽  
Genetic Interactions ◽  
Automated Image Analysis ◽  
C Elegans ◽  
Larval Stages ◽  
Lethal Mutations

ABSTRACTBackgroundThe advancement in automated image based microscopy platforms coupled with high throughput liquid workflows has facilitated the design of large scale screens utilizing multicellular model organisms such as Caenorhabditis elegans to identify genetic interactions, therapeutic drugs or disease modifiers. However, the analysis of essential genes has lagged behind because lethal or sterile mutations pose a bottleneck for high throughput approaches.ResultsIn C. elegans, non-conditional lethal mutations can be maintained in heterozygosis using chromosome balancers, commonly labelled with GFP in the pharynx. Moreover gene-expression is typically monitored by the use of fluorescent reporters marked with the same fluorophore. Therefore, the separation of the different populations of animals at early larval stages represents a challenge. Here, we develop a sorting strategy capable of selecting homozygous mutants carrying a GFP stress reporter from GFP-balanced animals at early larval stages. Because sorting is not completely error-free, we develop an automated high-throughput image-analysis protocol that identifies and discards animals carrying the chromosome balancer. We demonstrate the experimental usefulness of combining sorting of homozygous lethal mutants and automated image-analysis in a functional genomic RNAi screen for genes that genetically interact with mitochondrial prohibitin (PHB). Lack of PHB results in embryonic lethality, while, homozygous PHB deletion mutants develop into sterile adults due to maternal contribution and strongly induce the mitochondrial unfolded protein response (UPRmt). In a chromosome-wide RNAi screen for C. elegans genes having human orthologues, we uncover both, known and new PHB genetic interactors affecting the UPRmt and growth.ConclusionsA systematic way to analyse genetic interactions of essential genes in multicellular organisms is lacking. The method presented here allows the study of balanced lethal mutations in a high-throughput manner and can be easily adapted depending on the user’s requirements. Therefore, it will serve as a useful resource for the C. elegans community for probing new biological aspects of essential nematode genes as well as the generation of more comprehensive genetic networks.

Maternal-effect lethal mutations on linkage group II of Caenorhabditis elegans.

Genetics ◽  
1988 ◽  
Vol 120 (4) ◽  
pp. 977-986
Author(s):  
K J Kemphues ◽  
M Kusch ◽  
N Wolf
Keyword(s):  
Caenorhabditis Elegans ◽  
Linkage Group ◽  
Maternal Effect ◽  
Essential Genes ◽  
The Other ◽  
C Elegans ◽  
Lethal Mutations ◽  
Embryonic Lethal ◽  
Embryonic Lethal Mutations ◽  
F1 Progeny

Abstract We have analyzed a set of linkage group (LG) II maternal-effect lethal mutations in Caenorhabditis elegans isolated by a new screening procedure. Screens of 12,455 F1 progeny from mutagenized adults resulted in the recovery of 54 maternal-effect lethal mutations identifying 29 genes. Of the 54 mutations, 39 are strict maternal-effect mutations defining 17 genes. These 17 genes fall into two classes distinguished by frequency of mutation to strict maternal-effect lethality. The smaller class, comprised of four genes, mutated to strict maternal-effect lethality at a frequency close to 5 X 10(-4), a rate typical of essential genes in C. elegans. Two of these genes are expressed during oogenesis and required exclusively for embryogenesis (pure maternal genes), one appears to be required specifically for meiosis, and the fourth has a more complex pattern of expression. The other 13 genes were represented by only one or two strict maternal alleles each. Two of these are identical genes previously identified by nonmaternal embryonic lethal mutations. We interpret our results to mean that although many C. elegans genes can mutate to strict maternal-effect lethality, most genes mutate to that phenotype rarely. Pure maternal genes, however, are among a smaller class of genes that mutate to maternal-effect lethality at typical rates. If our interpretation is correct, we are near saturation for pure maternal genes in the region of LG II balanced by mnC1. We conclude that the number of pure maternal genes in C. elegans is small, being probably not much higher than 12.

scholarly journals Combined flow cytometry and high-throughput image analysis for the study of essential genes in Caenorhabditis elegans

BMC Biology ◽  
2018 ◽  
Vol 16 (1) ◽  
Author(s):  
Blanca Hernando-Rodríguez ◽  
Annmary Paul Erinjeri ◽  
María Jesús Rodríguez-Palero ◽  
Val Millar ◽  
Sara González-Hernández ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Image Analysis ◽  
Caenorhabditis Elegans ◽  
High Throughput ◽  
Essential Genes ◽  
Combined Flow

scholarly journals Insect-Derived Cecropins Display Activity against Acinetobacter baumannii in a Whole-Animal High-Throughput Caenorhabditis elegans Model

2015 ◽  
Vol 59 (3) ◽  
pp. 1728-1737 ◽  
Author(s):  
Elamparithi Jayamani ◽  
Rajmohan Rajamuthiah ◽  
Jonah Larkins-Ford ◽  
Beth Burgwyn Fuchs ◽  
Annie L. Conery ◽  
...  
Keyword(s):  
Caenorhabditis Elegans ◽  
Acinetobacter Baumannii ◽  
High Throughput ◽  
Sequence Data ◽  
Treatment Options ◽  
Automated Image Analysis ◽  
Screening Assay ◽  
Content Type ◽  
C Elegans ◽  
Cecropin A

ABSTRACTThe rise of multidrug-resistantAcinetobacter baumanniiand a concomitant decrease in antibiotic treatment options warrants a search for new classes of antibacterial agents. We have found thatA. baumanniiis pathogenic and lethal to the model host organismCaenorhabditis elegansand have exploited this phenomenon to develop an automated, high-throughput, high-content screening assay in liquid culture that can be used to identify novel antibiotics effective againstA. baumannii. The screening assay involves coincubatingC. eleganswithA. baumanniiin 384-well plates containing potential antibacterial compounds. At the end of the incubation period, worms are stained with a dye that stains only dead animals, and images are acquired using automated microscopy and then analyzed using an automated image analysis program. This robust assay yields a Z′ factor consistently greater than 0.7. In a pilot experiment to test the efficacy of the assay, we screened a small custom library of synthetic antimicrobial peptides (AMPs) that were synthesized using publicly available sequence data and/or transcriptomic data from immune-challenged insects. We identified cecropin A and 14 other cecropin or cecropin-like peptides that were able to enhanceC. eleganssurvival in the presence ofA. baumannii. Interestingly, one particular hit, BR003-cecropin A, a cationic peptide synthesized by the mosquitoAedes aegypti, showed antibiotic activity against a panel of Gram-negative bacteria and exhibited a low MIC (5 μg/ml) againstA. baumannii. BR003-cecropin A causes membrane permeability inA. baumannii, which could be the underlying mechanism of its lethality.

scholarly journals Automated and customizable quantitative image analysis of whole Caenorhabditis elegans germlines

Genetics ◽  
2021 ◽  
Author(s):  
Erik Toraason ◽  
Victoria L Adler ◽  
Nicole A Kurhanewicz ◽  
Acadia DiNardo ◽  
Adam M Saunders ◽  
...  
Keyword(s):  
Image Analysis ◽  
Caenorhabditis Elegans ◽  
Germ Cell ◽  
Meiotic Prophase ◽  
Quantitative Image Analysis ◽  
Dna Breaks ◽  
C Elegans ◽  
Quantitative Image ◽  
Cytological Features ◽  
Single Nucleus

Abstract Arranged in a spatial-temporal gradient for germ cell development, the adult germline of Caenorhabditis elegans is an excellent system for understanding the generation, differentiation, function, and maintenance of germ cells. Imaging whole C. elegans germlines along the distal-proximal axis enables powerful cytological analyses of germ cell nuclei as they progress from the pre-meiotic tip through all the stages of meiotic prophase I. To enable high-content image analysis of whole C. elegans gonads, we developed a custom algorithm and pipelines to function with image processing software that enables: (1) quantification of cytological features at single nucleus resolution from immunofluorescence images; and (2) assessment of these individual nuclei based on their position within the germline. We show the capability of our quantitative image analysis approach by analyzing multiple cytological features of meiotic nuclei in whole C. elegans germlines. First, we quantify double-strand DNA breaks (DSBs) per nucleus by analyzing DNA-associated foci of the recombinase RAD-51 at single-nucleus resolution in the context of whole germline progression. Second, we quantify the DSBs that are licensed for crossover repair by analyzing foci of MSH-5 and COSA-1 when they associate with the synaptonemal complex during meiotic prophase progression. Finally, we quantify P-granule composition across the whole germline by analyzing the colocalization of PGL-1 and ZNFX-1 foci. Our image analysis pipeline is an adaptable and useful method for researchers spanning multiple fields using the C. elegans germline as a model system.

Reporter gene constructs suggest that the Caenorhabditis elegans avermectin receptor beta-subunit is expressed solely in the pharynx.

1997 ◽  
Vol 200 (10) ◽  
pp. 1509-1514 ◽  
Author(s):  
D L Laughton ◽  
G G Lunt ◽  
A J Wolstenholme
Keyword(s):  
Caenorhabditis Elegans ◽  
Genomic Sequence ◽  
Gene Promoter ◽  
Pcr Amplification ◽  
Beta Subunit ◽  
Amino Acid Residues ◽  
C Elegans ◽  
Larval Stages ◽  
Major Mode ◽  
Flanking Sequences

Gene promoter/LacZ reporter constructs were made in order to analyse the expression of the beta-subunit of the Caenorhabditis elegans glutamate-gated Cl- channel (Glu-Cl) receptor. Southern blot analysis of the C. elegans cosmid C35E8 identified a 4kbp EcoRI fragment which contained the 5' portion of the Glu-Cl beta coding sequence together with 5' flanking sequences. This was subcloned and used as the template for polymerase chain reaction (PCR) amplification of a DNA fragment encoding the first 24 amino acid residues of Glu-Cl beta together with 1.4 kbp of 5' genomic sequence. The fragment was subcloned into the LacZ expression vector pPD22.11 to form a translational reporter fusion. After injection of the construct into worms, six stably transformed lines were established and assayed for beta-galactosidase activity. Stained nuclei were observed in the pharyngeal metacorpus in adults and in all larval stages, and stained nuclei were seen in many embryos undergoing morphogenesis. Additional stained nuclei towards the terminal bulb of the pharynx were observed in larval stages. These results provide further evidence that the Glu-Cl receptor mediates the glutamatergic inhibition of pharyngeal muscle via the M3 motor neurone and point to inhibition of pharyngeal pumping as a major mode of action for avermectins.

scholarly journals High-throughput microfluidic micropipette aspiration device to probe time-scale dependent nuclear mechanics in intact cells

Lab on a Chip ◽  
2019 ◽  
Vol 19 (21) ◽  
pp. 3652-3663 ◽  
Author(s):  
Patricia M. Davidson ◽  
Gregory R. Fedorchak ◽  
Solenne Mondésert-Deveraux ◽  
Emily S. Bell ◽  
Philipp Isermann ◽  
...  
Keyword(s):  
Image Analysis ◽  
High Throughput ◽  
Time Scale ◽  
Viscoelastic Properties ◽  
Micropipette Aspiration ◽  
Automated Image Analysis ◽  
Cell Nuclei ◽  
Intact Cells ◽  
Nuclear Mechanics ◽  
Analysis Platform

We report the development, validation, and application of an easy-to-use microfluidic micropipette aspiration device and automated image analysis platform that enables high-throughput measurements of the viscoelastic properties of cell nuclei.

scholarly journals CAENORHABDITIS ELEGANS AS A MODEL OF AIR POLLUTION TOXICITY DURING DEVELOPMENT AND LIFESPAN

2019 ◽  
Vol 3 (Supplement_1) ◽  
pp. S97-S97
Author(s):  
Amin Haghani ◽  
Hans M Dalton ◽  
Nikoo Safi ◽  
Farimah Shirmohammadi ◽  
Constantinos Sioutas ◽  
...  
Keyword(s):  
Air Pollution ◽  
Caenorhabditis Elegans ◽  
Mouse Models ◽  
High Throughput ◽  
Molecular Mechanisms ◽  
Cultured Cells ◽  
Exposure Model ◽  
C Elegans ◽  
Biological Responses ◽  
Short Term Exposure

Abstract Air pollution (AirPoll) is among the leading human mortality risk factors and yet little is known about the molecular mechanisms of this global environmental toxin. Our recent studies using mouse models even showed genetic variation and sex can alter biological responses to air pollution. To expand genetic studies of AirPoll toxicity throughout the lifespan, we introduced Caenorhabditis elegans as a new AirPoll exposure model which has a short lifespan, high throughput capabilities and shared longevity pathways with mammals. Acute exposure of C. elegans to airborne nanosized AirPoll matter (nPM) caused similar gene expression changes to our prior findings in cell culture and mouse models. Initial C. elegans responses to nPM included antioxidant, inflammatory and Alzheimer homolog genes. The magnitude of changes was dependent on the developmental stage of the worms. Even short term exposure of C. elegans to nPM altered developmental and lifespan hormetic effects, with pathways that included skn-1/Nrf family antioxidant responses. We propose C. elegans as a new and complementary model for mouse and cultured cells to study AirPoll across the lifespan. Future chronic nPM exposure and high throughput genetic screening of C. elegans can identify other major regulators of the developmental and lifespan effects of air pollution. This work was supported by grants R01AG051521 (CEF); R21AG05020 (CEF); Cure Alzheimer’s Fund (CEF); R01GM109028 (SPC), F31AG051382 (HMD) and T32AG000037 (HMD), T32AG052374 (AH).

scholarly journals Altered expression of an L1-specific, O-linked cuticle surface glycoprotein in mutants of the nematode Caenorhabditis elegans.

1991 ◽  
Vol 115 (5) ◽  
pp. 1237-1247 ◽  
Author(s):  
R M Hemmer ◽  
S G Donkin ◽  
K J Chin ◽  
D G Grenache ◽  
H Bhatt ◽  
...  
Keyword(s):  
Caenorhabditis Elegans ◽  
Specific Antigen ◽  
Surface Glycoprotein ◽  
Wild Type ◽  
Nematode Caenorhabditis Elegans ◽  
Altered Expression ◽  
C Elegans ◽  
Larval Stages ◽  
Sds Page ◽  
Free Living Nematode

Mouse mAb M38 was used in indirect immunofluorescence experiments to detect a stage-specific antigen on the surface of the first larval stage (L1) of the free-living nematode Caenorhabditis elegans, and to detect alterations in the apparent expression of this antigen in two distinct classes of C. elegans mutants. In previously described srf-2 and srf-3 mutants (Politz S. M., M. T. Philipp, M. Estevez, P.J. O'Brien, and K. J. Chin. 1990. Proc. Natl. Acad. Sci. USA. 87:2901-2905), the antigen is not detected on the surface of any stage. Conversely, in srf-(yj43) and other similar mutants, the antigen is expressed on the surface of the first through the fourth (L4) larval stages. To understand the molecular basis of these alterations, the antigen was characterized in gel immunoblotting experiments. After SDS-PAGE separation and transfer to nitrocellulose, M38 detected a protein antigen in extracts of wild-type L1 populations. The antigen was sensitive to digestion by Pronase and O-glycanase (endo-alpha-N-acetylgalactosaminidase), suggesting that it is an O-linked glycoprotein. This antigen was not detected in corresponding extracts of wild-type L4s or srf-2 or srf-3 L1s, but was detected in extracts of srf-(yj43) L4s. The antigen-defective phenotype of srf-3 was epistatic to the heterochronic mutant phenotype of srf-(yj43) in immunofluorescence tests of the srf-3 srf-(yj43) double mutant, suggesting that srf-(yj43) causes incorrect regulation of a pathway of antigen formation that requires wild-type srf-3 activity.

Sign in / Sign up

Export Citation Format

Share Document