The PAX gene egl-38 mediates developmental patterning in Caenorhabditis elegans

Development ◽  
1997 ◽  
Vol 124 (20) ◽  
pp. 3919-3928 ◽  
Author(s):  
H.M. Chamberlin ◽  
R.E. Palmer ◽  
A.P. Newman ◽  
P.W. Sternberg ◽  
D.L. Baillie ◽  
...  
Keyword(s):  
Transcription Factors ◽  
Genetic Analysis ◽  
Molecular Genetic ◽  
Egg Laying ◽  
Paired Domain ◽  
C Elegans ◽  
Developmental Patterning ◽  
Pax Family ◽  
Insight Into

Mutations in the C. elegans gene egl-38 result in a discrete set of defects in developmental pattern formation. In the developing egg-laying system of egl-38 mutant hermaphrodites, the identity of four uterine cells is disrupted and they adopt the fate of their neighbor cells. Likewise, the identity of two rectal epithelial cells in the male tail is disrupted and one of these cells adopts the fate of its neighbor cell. Genetic analysis suggests that the egl-38 functions in the tail and the egg-laying system are partially separable, as different egl-38 mutations can preferentially disrupt the different functions. We have cloned egl-38 and shown that it is a member of the PAX family of genes, which encode transcription factors implicated in a variety of developmental patterning events. The predicted EGL-38 protein is most similar to the mammalian class of proteins that includes PAX2, PAX5 and PAX8. The sequence of egl-38 mutant DNA indicates that the tissue-preferential defects of egl-38 mutations result from substitutions in the DNA-binding paired domain of the EGL-38 protein. egl-38 thus provides the first molecular genetic insight into two specific patterning events that occur during C. elegans development and also provides the opportunity to investigate the in vivo functions of this class of PAX proteins with single cell resolution.

scholarly journals The neural G protein Gαo tagged with GFP at an internal loop is functional in C. elegans

2021 ◽  
Author(s):  
Santosh Kumar ◽  
Andrew C Olson ◽  
Michael R Koelle
Keyword(s):  
Genetic Analysis ◽  
G Protein ◽  
Molecular Mechanisms ◽  
Fluorescent Protein ◽  
Protein Complexes ◽  
Egg Laying ◽  
Biochemical Purification ◽  
Internal Loop ◽  
C Elegans

Abstract Gαo is the alpha subunit of the major heterotrimeric G protein in neurons and mediates signaling by every known neurotransmitter, yet the signaling mechanisms activated by Gαo remain to be fully elucidated. Genetic analysis in Caenorhabditis elegans has shown that Gαo signaling inhibits neuronal activity and neurotransmitter release, but studies of the molecular mechanisms underlying these effects have been limited by lack of tools to complement genetic studies with other experimental approaches. Here we demonstrate that inserting the green fluorescent protein (GFP) into an internal loop of the Gαo protein results in a tagged protein that is functional in vivo and that facilitates cell biological and biochemical studies of Gαo. Transgenic expression of Gαo-GFP rescues the defects caused by loss of endogenous Gαo in assays of egg laying and locomotion behaviors. Defects in body morphology caused by loss of Gαo are also rescued by Gαo-GFP. The Gαo-GFP protein is localized to the plasma membrane of neurons, mimicking localization of endogenous Gαo. Using GFP as an epitope tag, Gαo-GFP can be immunoprecipitated from C. elegans lysates to purify Gαo protein complexes. The Gαo-GFP transgene reported in this study enables studies involving in vivo localization and biochemical purification of Gαo to complement the already well-developed genetic analysis of Gαo signaling.

scholarly journals The neural G protein Gαo tagged with GFP at an internal loop is functional in C. elegans

2021 ◽  
Author(s):  
Santosh Kumar ◽  
Andrew C. Olson ◽  
Michael R. Koelle
Keyword(s):  
Genetic Analysis ◽  
G Protein ◽  
Molecular Mechanisms ◽  
Fluorescent Protein ◽  
Protein Complexes ◽  
Egg Laying ◽  
Biochemical Purification ◽  
Internal Loop ◽  
C Elegans

AbstractGαo is the alpha subunit of the major heterotrimeric G protein in neurons and mediates signaling by every known neurotransmitter, yet the signaling mechanisms activated by Gαo remain to be fully elucidated. Genetic analysis in Caenorhabditis elegans has shown that Gαo signaling inhibits neuronal activity and neurotransmitter release, but studies of the molecular mechanisms underlying these effects have been limited by lack of tools to complement genetic studies with other experimental approaches. Here we demonstrate that inserting the green fluorescent protein (GFP) into an internal loop of the Gαo protein results in a tagged protein that is functional in vivo and that facilitates cell biological and biochemical studies of Gαo. Transgenic expression of Gαo-GFP rescues the defects caused by loss of endogenous Gαo in assays of egg laying and locomotion behaviors. Defects in body morphology caused by loss of Gαo are also rescued by Gαo-GFP. The Gαo-GFP protein is localized to the plasma membrane of neurons, mimicking localization of endogenous Gαo. Using GFP as an epitope tag, Gαo-GFP can be immunoprecipitated from C. elegans lysates to purify Gαo protein complexes. The Gαo-GFP transgene reported in this study enables studies involving in vivo localization and biochemical purification of Gαo to complement the already well-developed genetic analysis of Gαo signaling.

scholarly journals Microtubule-dependent ribosome localization in C. elegans neurons

eLife ◽  
2017 ◽  
Vol 6 ◽  
Author(s):  
Kentaro Noma ◽  
Alexandr Goncharov ◽  
Mark H Ellisman ◽  
Yishi Jin
Keyword(s):  
Cell Biology ◽  
Neuronal Development ◽  
Ultrastructural Level ◽  
Synthesis Methods ◽  
Tissue Specific ◽  
C Elegans ◽  
Multicellular Organisms ◽  
Axonal Trafficking ◽  
Insight Into

Subcellular localization of ribosomes defines the location and capacity for protein synthesis. Methods for in vivo visualizing ribosomes in multicellular organisms are desirable in mechanistic investigations of the cell biology of ribosome dynamics. Here, we developed an approach using split GFP for tissue-specific visualization of ribosomes in Caenorhabditis elegans. Labeled ribosomes are detected as fluorescent puncta in the axons and synaptic terminals of specific neuron types, correlating with ribosome distribution at the ultrastructural level. We found that axonal ribosomes change localization during neuronal development and after axonal injury. By examining mutants affecting axonal trafficking and performing a forward genetic screen, we showed that the microtubule cytoskeleton and the JIP3 protein UNC-16 exert distinct effects on localization of axonal and somatic ribosomes. Our data demonstrate the utility of tissue-specific visualization of ribosomes in vivo, and provide insight into the mechanisms of active regulation of ribosome localization in neurons.

scholarly journals Occurrence of Salmonella typhimurium resistance under sublethal/repeated exposure to cauliflower infusion and infection effects on Caernohabditis elegans host test organism

2019 ◽  
Vol 26 (2) ◽  
pp. 151-159
Author(s):  
Maria Sanz-Puig ◽  
Alejandra Arana-Lozano ◽  
M Consuelo Pina-Pérez ◽  
Pablo Fernández ◽  
Antonio Martínez ◽  
...  
Keyword(s):  
Food Industry ◽  
Research Work ◽  
Test Organism ◽  
Egg Laying ◽  
Repeated Exposure ◽  
Resistant Bacteria ◽  
Bacterial Cells ◽  
Natural Antimicrobial ◽  
C Elegans

Resistant bacteria to antimicrobials are increasingly emerging in medical, food industry and livestock environments. The present research work assesses the capability of Salmonella enterica var Typhimurium to become adapted under the exposure to a natural cauliflower antimicrobial by-product infusion in consecutive repeated exposure cycles. Caenorhabditis elegans was proposed as in vivo host-test organism to compare possible changes in the virulent pattern of the different rounds treated S. enterica var Typhimurium and untreated bacterial cells. According to the obtained results, S. enterica var Typhimurium was able to generate resistance against a repeated exposure to cauliflower by-product infusion 5% (w/v), increasing the resistance with the number of exposed repetitions. Meanwhile, at the first exposure, cauliflower by-product infusion was effective in reducing S. enterica var Typhimurium (≈1 log10 cycle), and S. enterica var Typhimurium became resistant to this natural antimicrobial after the second and third treatment-round and was able to grow (≈1 log10 cycle). In spite of the increased resistance observed for repeatedly treated bacteria, the present study reveals no changes on C. elegans infection effects between resistant and untreated S. enterica var Typhimurium, according to phenotypic parameters evaluation (lifespan duration and egg-laying).

scholarly journals Microfluidic Electric Egg-Laying Assay and Application to In-vivo Toxicity Screening of Microplastics using C. elegans

2021 ◽  
Author(s):  
Khaled Youssef ◽  
Daphne Archonta ◽  
Terrance J. Kubiseseki ◽  
Anurag Tandon ◽  
Pouya Rezai
Keyword(s):  
Environmental Pollutants ◽  
Egg Laying ◽  
Electric Signal ◽  
Data Sets ◽  
Toxicity Screening ◽  
C Elegans ◽  
Toxicological Studies ◽  
Individual Worm ◽  
Polystyrene Microparticles

AbstractEnvironmental pollutants like microplastics are posing health concerns on aquatic animals and the ecosystem. Microplastic toxicity studies using C. elegans as a model are evolving but methodologically hindered from obtaining statistically strong data sets, detecting toxicity effects based on microplastics uptake, and correlating physiological and behavioural effects at an individual-worm level. In this paper, we report a novel microfluidic electric egg-laying assay for phenotypical assessment of multiple worms in parallel. The effects of glucose and polystyrene microplastics at various concentrations on the worms’ electric egg-laying, length, diameter, and length contraction during exposure to electric signal were studied. The device contained eight parallel worm-dwelling microchannels called electric traps, with equivalent electrical fields, in which the worms were electrically stimulated for egg deposition and fluorescently imaged for assessment of neuronal and microplastic uptake expression. A new bidirectional stimulation technique was developed, and the device design was optimized to achieve a testing efficiency of 91.25%. Exposure of worms to 100mM glucose resulted in a significant reduction in their egg-laying and size. The effects of 1μm polystyrene microparticles at concentrations of 100 and 1000 mg/L on the electric egg-laying behaviour, size, and neurodegeneration of N2 and NW1229 (expressing GFP pan-neuronally) worms were also studied. Of the two concentrations, 1000 mg/L caused severe egg-laying deficiency and growth retardation as well as neurodegeneration. Additionally, using single-worm level phenotyping, we noticed intra-population variability in microplastics uptake and correlation with the above physiological and behavioural phenotypes, which was hidden in the population-averaged results. Taken together, these results suggest the appropriateness of our microfluidic assay for toxicological studies and for assessing the phenotypical heterogeneity in response to microplastics.

scholarly journals Acentrosomal spindle assembly and stability in C. elegans oocytes requires a kinesin-12 non-motor microtubule interaction domain

2021 ◽  
Author(s):  
Ian Daniel Wolff ◽  
Jeremy Alden Hollis ◽  
Sarah Marie Wignall
Keyword(s):  
Adaptor Protein ◽  
Direct Interaction ◽  
Spindle Assembly ◽  
Meiotic Spindle ◽  
Interaction Domain ◽  
Microtubule Binding ◽  
C Elegans ◽  
Insight Into

During the meiotic divisions in oocytes, microtubules are sorted and organized by motor proteins to generate a bipolar spindle in the absence of centrosomes. In most organisms, kinesin-5 family members crosslink and slide microtubules to generate outward force that promotes acentrosomal spindle bipolarity. However, the mechanistic basis for how other kinesin families act on acentrosomal spindles has not been explored. We investigated this question in C. elegans oocytes, where kinesin-5 is not required to generate outward force. Instead, the kinesin-12 family motor KLP-18 performs this function. KLP-18 acts with adaptor protein MESP-1 (meiotic spindle 1) to sort microtubule minus ends to the periphery of a microtubule array, where they coalesce into spindle poles. If either of these proteins is depleted, outward sorting of microtubules is lost and minus ends converge to form a monoaster. Here we use a combination of in vitro biochemical assays and in vivo mutant analysis to provide insight into the mechanism by which these proteins collaborate to promote acentrosomal spindle assembly. We identify a microtubule binding site on the C-terminal stalk of KLP-18 and demonstrate that a direct interaction between the KLP-18 stalk and MESP-1 activates non-motor microtubule binding. We also provide evidence that this C-terminal domain is required for KLP-18 activity during spindle assembly and show that KLP-18 is continuously required to maintain spindle bipolarity. This study thus provides new insight into the construction and maintenance of the oocyte acentrosomal spindle as well as into kinesin-12 mechanism and regulation.

scholarly journals Interpreting TMEM67 missense variants of uncertain significance (VUS) in an animal model

2021 ◽  
Author(s):  
Karen I Lange ◽  
Sunayna Best ◽  
Sofia Tsiropoulou ◽  
Ian Berry ◽  
Colin A Johnson ◽  
...  
Keyword(s):  
Animal Model ◽  
Molecular Genetic ◽  
Genetic Diagnosis ◽  
Knock Out ◽  
Variants Of Uncertain Significance ◽  
C Elegans ◽  
In Vivo Animal Model ◽  
Uncertain Significance

Purpose: A molecular genetic diagnosis is essential for accurate counselling and management of patients with ciliopathies. Uncharacterized missense alleles are often classified as variants of uncertain significance (VUS) and are not clinically useful. In this study, we explore the use of a tractable animal model (C. elegans) for in vivo interpretation of missense VUS alleles of TMEM67, a gene frequently mutated as a cause of ciliopathies. Methods: CRISPR/Cas9 gene editing was used to generate homozygous worm strains carrying TMEM67 patient variants. Quantitative phenotypic assays (dye filling, roaming, chemotaxis) assessed cilia structure and function. Results were validated by genetic complementation assays in a human TMEM67 knock-out hTERT-RPE1 cell line. Results: Quantitative assays in C. elegans distinguished between known benign (Asp359Glu, Thr360Ala) and pathogenic (Glu361Ter, Gln376Pro) variants. Analysis of seven missense VUS alleles predicted two benign (Cys173Arg, Thr176Ile) and four pathogenic variants (Cys170Tyr, His782Arg, Gly786Glu, His790Arg). Results from one VUS (Gly979Arg) were inconclusive in worms, but additional in vitro validation suggested it was likely benign. Conclusion: Efficient genome editing and quantitative functional assays in C. elegans make it a tractable in vivo animal model that allows stratification and rapid, cost-effective interpretation of ciliopathy-associated missense VUS alleles.

scholarly journals Interplay of RFX transcription factors 1, 2 and 3 in motile ciliogenesis

2020 ◽  
Vol 48 (16) ◽  
pp. 9019-9036
Author(s):  
Sylvain Lemeille ◽  
Marie Paschaki ◽  
Dominique Baas ◽  
Laurette Morlé ◽  
Jean-Luc Duteyrat ◽  
...  
Keyword(s):  
Transcription Factors ◽  
Transcriptional Control ◽  
Ex Vivo ◽  
Ependymal Cells ◽  
Clear Understanding ◽  
Animal Development ◽  
C Elegans ◽  
And Function ◽  
Redundant Functions

Abstract Cilia assembly is under strict transcriptional control during animal development. In vertebrates, a hierarchy of transcription factors (TFs) are involved in controlling the specification, differentiation and function of multiciliated epithelia. RFX TFs play key functions in the control of ciliogenesis in animals. Whereas only one RFX factor regulates ciliogenesis in C. elegans, several distinct RFX factors have been implicated in this process in vertebrates. However, a clear understanding of the specific and redundant functions of different RFX factors in ciliated cells remains lacking. Using RNA-seq and ChIP-seq approaches we identified genes regulated directly and indirectly by RFX1, RFX2 and RFX3 in mouse ependymal cells. We show that these three TFs have both redundant and specific functions in ependymal cells. Whereas RFX1, RFX2 and RFX3 occupy many shared genomic loci, only RFX2 and RFX3 play a prominent and redundant function in the control of motile ciliogenesis in mice. Our results provide a valuable list of candidate ciliary genes. They also reveal stunning differences between compensatory processes operating in vivo and ex vivo.

scholarly journals CSC-1

2003 ◽  
Vol 161 (2) ◽  
pp. 229-236 ◽  
Author(s):  
Alper Romano ◽  
Annika Guse ◽  
Ivica Krascenicova ◽  
Heinke Schnabel ◽  
Ralf Schnabel ◽  
...  
Keyword(s):  
Caenorhabditis Elegans ◽  
Genetic Analysis ◽  
Chromosome Segregation ◽  
Kinase Activity ◽  
Aurora B ◽  
Aurora B Kinase ◽  
C Elegans ◽  
The Individual

The Aurora B kinase complex is a critical regulator of chromosome segregation and cytokinesis. In Caenorhabditis elegans, AIR-2 (Aurora B) function requires ICP-1 (Incenp) and BIR-1 (Survivin). In various systems, Aurora B binds to orthologues of these proteins. Through genetic analysis, we have identified a new subunit of the Aurora B kinase complex, CSC-1. C. elegans embryos depleted of CSC-1, AIR-2, ICP-1, or BIR-1 have identical phenotypes. CSC-1, BIR-1, and ICP-1 are interdependent for their localization, and all are required for AIR-2 localization. In vitro, CSC-1 binds directly to BIR-1. The CSC-1/BIR-1 complex, but not the individual subunits, associates with ICP-1. CSC-1 associates with ICP-1, BIR-1, and AIR-2 in vivo. ICP-1 dramatically stimulates AIR-2 kinase activity. This activity is not stimulated by CSC-1/BIR-1, suggesting that these two subunits function as targeting subunits for AIR-2 kinase.

Sign in / Sign up

Export Citation Format

Share Document