Identification of Caenorhabditis elegans Genes Required for Neuronal Differentiation and Migration

Genetics ◽  
1998 ◽  
Vol 148 (1) ◽  
pp. 151-165 ◽  
Author(s):  
Wayne C Forrester ◽  
Elliot Perens ◽  
Jennifer A Zallen ◽  
Gian Garriga
Keyword(s):  
Caenorhabditis Elegans ◽  
Neuronal Differentiation ◽  
Homeodomain Protein ◽  
C Elegans ◽  
Differentiation Marker ◽  
And Migration ◽  
Null Mutants ◽  
Migrating Cells

Abstract To understand the mechanisms that guide migrating cells, we have been studying the embryonic migrations of the C. elegans canal-associated neurons (CANs). Here, we describe two screens used to identify genes involved in CAN migration. First, we screened for mutants that died as clear larvae (Clr) or had withered tails (Wit), phenotypes displayed by animals lacking normal CAN function. Second, we screened directly for mutants with missing or misplaced CANs. We isolated and characterized 30 mutants that defined 14 genes necessary for CAN migration. We found that one of the genes, ceh-10, specifies CAN fate. ceh-10 had been defined molecularly as encoding a homeodomain protein expressed in the CANs. Mutations that reduce ceh-10 function result in Wit animals with CANs that are partially defective in their migrations. Mutations that eliminate ceh-10 function result in Clr animals with CANs that fail to migrate or express CEH-23, a CAN differentiation marker. Null mutants also fail to express CEH-10, suggesting that CEH-10 regulates its own expression. Finally, we found that ceh-10 is necessary for the differentiation of AIY and RMED, two additional cells that express CEH-10.

scholarly journals An efficient genome editing strategy to generate putative null mutants in Caenorhabditis elegans using CRISPR/Cas9

2018 ◽  
Author(s):  
Han Wang ◽  
Heenam Park ◽  
Jonathan Liu ◽  
Paul W. Sternberg
Keyword(s):  
Dna Repair ◽  
Caenorhabditis Elegans ◽  
Genome Editing ◽  
Gene Function ◽  
Unique Sequence ◽  
Target Site ◽  
Null Alleles ◽  
Wild Type ◽  
C Elegans ◽  
Null Mutants

AbstractNull mutants are essential for analyzing gene function. Here, we describe a simple and efficient method to generate Caenorhabditis elegans null mutants using CRISPR/Cas9 and short single stranded DNA oligo repair templates to insert a universal 43-nucleotide-long stop knock-in (STOP-IN) cassette into the early exons of target genes. This cassette has stop codons in all three reading frames and leads to frameshifts, which will generate putative null mutations regardless of the reading frame of the insertion position in exons. The STOP-IN cassette also contains an exogenous Cas9 target site that allows further genome editing and provides a unique sequence that simplifies the identification of successful insertion events via PCR. As a proof of concept, we inserted the STOP-IN cassette right at a Cas9 target site in aex-2 to generate new putative null alleles by injecting preassembled Cas9 ribonucleoprotein and a short synthetic single stranded DNA repair template containing the STOP-IN cassette and two 35-nucleotide-long homology arms identical to the sequences flanking the Cas9 cut site. We showed that these new aex-2 alleles phenocopied an existing loss-of-function allele of aex-2. We further showed that the new aex-2 null alleles could be reverted back to the wild-type sequence by targeting exogenous Cas9 cut site included in the STOP-IN cassette and providing a single stranded wild-type DNA repair oligo. We applied our STOP-IN method to generate new putative null mutants for additional 20 genes, including three pharyngeal muscle-specific genes (clik-1, clik-2, and clik-3), and reported a high insertion rate (46%) based on the animals we screened. We showed that null mutations of clik-2 cause recessive lethality with a severe pumping defect and clik-3 null mutants have a mild pumping defect, while clik-1 is dispensable for pumping. We expect that the knock-in method using the STOP-IN cassette will facilitate the generation of new null mutants to understand gene function in C. elegans and other genetic model organisms.SummaryWe report a simple and efficient CRISPR/Cas9 genome editing strategy to generate putative null C. elegans mutants by inserting a small universal stop knock-in (STOP-IN) cassette with stop codons in three frames and frameshifts. The strategy is cloning-free, with the mixture consisting of preassembled Cas9 ribonucleoprotein and single stranded repair DNA oligos directly injected into gonads of adult C. elegans. The universal STOP-IN cassette also contains a unique sequence that simplifies detection of successful knock-in events via PCR and an exogenous Cas9 target sequence that allows further genome editing.

Synergistic roles of homeodomain proteins UNC-62 homothorax and MLS-2 HMX/NKX in the specification of olfactory neurons in Caenorhabditis elegans

Genetics ◽  
2021 ◽  
Author(s):  
Yi-Wen Hsieh ◽  
Rui Xiong ◽  
Chiou-Fen Chuang
Keyword(s):  
Transcription Factor ◽  
Caenorhabditis Elegans ◽  
Incomplete Penetrance ◽  
Homeodomain Protein ◽  
Homeodomain Proteins ◽  
Complete Penetrance ◽  
Late Embryogenesis ◽  
Tale Homeodomain ◽  
Null Mutants

Abstract General identity of the Caenorhabditis elegans AWC olfactory neuron pair is specified by the OTX/OTD transcription factor CEH-36 and the HMG-box transcription factor SOX-2, followed by asymmetrical differentiation of the pair into two distinct subtypes, default AWCOFF and induced AWCON, through a stochastic signaling event. The HMX/NKX transcription factor MLS-2 regulates the expression of ceh-36 to specify general AWC identity. However, general AWC identity is lost in only one of the two AWC cells in the majority of mls-2 null mutants displaying defective general AWC identity, suggesting that additional transcription factors have a partially overlapping role with MLS-2 in the specification of general AWC identity. Here we identify a role of unc-62, encoding a homothorax/Meis/TALE homeodomain protein, in the specification of general AWC identity. As in mls-2 null mutants, unc-62 null mutants showed an incomplete penetrance in loss of general AWC identity. However, unc-62; mls-2 double mutants display a nearly complete penetrance of identity loss in both AWC cells. Thus, unc-62 and mls-2 have a partially overlapping function in the specification of general AWC identity. Furthermore, our genetic results suggest that mls-2 and unc-62 act cell autonomously in promoting the AWCON subtype. Together, our findings reveal the sequential roles of the unc-62 and mls-2 pair in AWC development, specification of general AWC identity in early embryogenesis and asymmetric differentiation of AWC subtypes in late embryogenesis.

scholarly journals PTRN-1, a microtubule minus end-binding CAMSAP homolog, promotes microtubule function in Caenorhabditis elegans neurons

eLife ◽  
2014 ◽  
Vol 3 ◽  
Author(s):  
Claire E Richardson ◽  
Kerri A Spilker ◽  
Juan G Cueva ◽  
John Perrino ◽  
Miriam B Goodman ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Caenorhabditis Elegans ◽  
Fluorescence Microscopy ◽  
Molecular Motors ◽  
Molecular Mechanisms ◽  
C Elegans ◽  
Structural Support ◽  
Neuronal Processes ◽  
Microtubule Function ◽  
Null Mutants

In neuronal processes, microtubules (MTs) provide structural support and serve as tracks for molecular motors. While it is known that neuronal MTs are more stable than MTs in non-neuronal cells, the molecular mechanisms underlying this stability are not fully understood. In this study, we used live fluorescence microscopy to show that the C. elegans CAMSAP protein PTRN-1 localizes to puncta along neuronal processes, stabilizes MT foci, and promotes MT polymerization in neurites. Electron microscopy revealed that ptrn-1 null mutants have fewer MTs and abnormal MT organization in the PLM neuron. Animals grown with a MT depolymerizing drug caused synthetic defects in neurite branching in the absence of ptrn-1 function, indicating that PTRN-1 promotes MT stability. Further, ptrn-1 null mutants exhibited aberrant neurite morphology and synaptic vesicle localization that is partially dependent on dlk-1. Our results suggest that PTRN-1 represents an important mechanism for promoting MT stability in neurons.

scholarly journals Evaluation of the effect of life extension of DHEA extract from sweet potato on Caenorhabditis elegans

2020 ◽  
Author(s):  
Huan Chen ◽  
Dan Zou ◽  
Lufeng Wang ◽  
Junyi Huang
Keyword(s):  
Caenorhabditis Elegans ◽  
Sweet Potato ◽  
Life Extension ◽  
E Coli ◽  
C Elegans ◽  
Nuclear Expression ◽  
Pump Rate ◽  
Extended Lifespan ◽  
Summary Statement ◽  
And Migration

AbstractProducts from natural sources are being used from centuries. This study investigates the potential antiaging activity of DHEA extracts from sweet potato. DHEA was extracted with the assistance of acid hydrolysis-ultrasonic, and the Caenorhabditis elegans model was used to investigate the antiaging activity. The results from the current study articulated that DHEA from sweet potato in 50 μmol / L effectively prolonged the life-span of C. elegans by 13.60%, but the effect was not concentration-dependent. Besides, DHEA had no effect on the growth of E. coli OP50 and the nematode pharyngeal pump rate, indicating that DHEA didn’t delay the aging of the nematode through calorie restriction. Further experiments demonstrated that DHEA would slow the growth of nematode body size, reduce the accumulation of ROS and lipofuscin of C. elegans. The expression and migration of daf-16 into the nucleus of nematode were significantly improved as well. The antiaging effect of DHEA on C. elegans may be achieved by strengthening the nematode’s ability to resist oxidative stress and promoting nuclear expression of the daf-16 gene.Summary StatementThis study proved that DHEA from sweet potato can extended lifespan of Caenorhabditis elegans induced by strengthening the antioxidant capacity and promoting the nuclear expression of daf-16

The glycomes of Caenorhabditis elegans and other model organisms

2002 ◽  
Vol 69 ◽  
pp. 117-134 ◽  
Author(s):  
Stuart M. Haslam ◽  
David Gems ◽  
Howard R. Morris ◽  
Anne Dell
Keyword(s):  
Caenorhabditis Elegans ◽  
Current Knowledge ◽  
Structural Data ◽  
Model Organisms ◽  
Entire Genome ◽  
C Elegans ◽  
The Face ◽  
Area Of Concern ◽  
Nematode Worm

There is no doubt that the immense amount of information that is being generated by the initial sequencing and secondary interrogation of various genomes will change the face of glycobiological research. However, a major area of concern is that detailed structural knowledge of the ultimate products of genes that are identified as being involved in glycoconjugate biosynthesis is still limited. This is illustrated clearly by the nematode worm Caenorhabditis elegans, which was the first multicellular organism to have its entire genome sequenced. To date, only limited structural data on the glycosylated molecules of this organism have been reported. Our laboratory is addressing this problem by performing detailed MS structural characterization of the N-linked glycans of C. elegans; high-mannose structures dominate, with only minor amounts of complex-type structures. Novel, highly fucosylated truncated structures are also present which are difucosylated on the proximal N-acetylglucosamine of the chitobiose core as well as containing unusual Fucα1–2Gal1–2Man as peripheral structures. The implications of these results in terms of the identification of ligands for genomically predicted lectins and potential glycosyltransferases are discussed in this chapter. Current knowledge on the glycomes of other model organisms such as Dictyostelium discoideum, Saccharomyces cerevisiae and Drosophila melanogaster is also discussed briefly.

The Effect of Mianserin on Lifespan of Caenorhabditis elegans is Abolished by Glucose

2021 ◽  
Vol 13 ◽  
Author(s):  
Abdullah Almotayri ◽  
Jency Thomas ◽  
Mihiri Munasinghe ◽  
Markandeya Jois
Keyword(s):  
Caenorhabditis Elegans ◽  
Scaffolding Protein ◽  
Lifespan Extension ◽  
Wild Type ◽  
E Coli ◽  
C Elegans ◽  
Risk Of Death ◽  
Increased Risk ◽  
Antidepressant Use ◽  
Extension Effect

Background: The antidepressant mianserin has been shown to extend the lifespan of Caenorhabditis elegans (C. elegans), a well-established model organism used in aging research. The extension of lifespan in C. elegans was shown to be dependent on increased expression of the scaffolding protein (ANK3/unc-44). In contrast, antidepressant use in humans is associated with an increased risk of death. The C. elegans in the laboratory are fed Escherichia coli (E. coli), a diet high in protein and low in carbohydrate, whereas a typical human diet is high in carbohydrates. We hypothesized that dietary carbohydrates might mitigate the lifespan-extension effect of mianserin. Objective: To investigate the effect of glucose added to the diet of C. elegans on the lifespan-extension effect of mianserin. Methods: Wild-type Bristol N2 and ANK3/unc-44 inactivating mutants were cultured on agar plates containing nematode growth medium and fed E. coli. Treatment groups included (C) control, (M50) 50 μM mianserin, (G) 73 mM glucose, and (M50G) 50 μM mianserin and 73 mM glucose. Lifespan was determined by monitoring the worms until they died. Statistical analysis was performed using the Kaplan-Meier version of the log-rank test. Results: Mianserin treatment resulted in a 12% increase in lifespan (P<0.05) of wild-type Bristol N2 worms but reduced lifespan by 6% in ANK3/unc-44 mutants, consistent with previous research. The addition of glucose to the diet reduced the lifespan of both strains of worms and abolished the lifespan-extension by mianserin. Conclusion: The addition of glucose to the diet of C. elegans abolishes the lifespan-extension effects of mianserin.

scholarly journals Functional Redundancy of the B9 Proteins and Nephrocystins in Caenorhabditis elegans Ciliogenesis

2008 ◽  
Vol 19 (5) ◽  
pp. 2154-2168 ◽  
Author(s):  
Corey L. Williams ◽  
Marlene E. Winkelbauer ◽  
Jenny C. Schafer ◽  
Edward J. Michaud ◽  
Bradley K. Yoder
Keyword(s):  
Caenorhabditis Elegans ◽  
Joubert Syndrome ◽  
Cystic Kidney ◽  
Basal Bodies ◽  
The Novel ◽  
C Elegans ◽  
Human Genes ◽  
Cilia Formation ◽  
Strong Candidate ◽  
Candidate Loci

Meckel-Gruber syndrome (MKS), nephronophthisis (NPHP), and Joubert syndrome (JBTS) are a group of heterogeneous cystic kidney disorders with partially overlapping loci. Many of the proteins associated with these diseases interact and localize to cilia and/or basal bodies. One of these proteins is MKS1, which is disrupted in some MKS patients and contains a B9 motif of unknown function that is found in two other mammalian proteins, B9D2 and B9D1. Caenorhabditis elegans also has three B9 proteins: XBX-7 (MKS1), TZA-1 (B9D2), and TZA-2 (B9D1). Herein, we report that the C. elegans B9 proteins form a complex that localizes to the base of cilia. Mutations in the B9 genes do not overtly affect cilia formation unless they are in combination with a mutation in nph-1 or nph-4, the homologues of human genes (NPHP1 and NPHP4, respectively) that are mutated in some NPHP patients. Our data indicate that the B9 proteins function redundantly with the nephrocystins to regulate the formation and/or maintenance of cilia and dendrites in the amphid and phasmid ciliated sensory neurons. Together, these data suggest that the human homologues of the novel B9 genes B9D2 and B9D1 will be strong candidate loci for pathologies in human MKS, NPHP, and JBTS.

scholarly journals Characterization of Caenorhabditis elegans Homologs of the Down Syndrome Candidate Gene DYRK1A

Genetics ◽  
2003 ◽  
Vol 163 (2) ◽  
pp. 571-580 ◽  
Author(s):  
William B Raich ◽  
Celine Moorman ◽  
Clay O Lacefield ◽  
Jonah Lehrer ◽  
Dusan Bartsch ◽  
...  
Keyword(s):  
Down Syndrome ◽  
Caenorhabditis Elegans ◽  
Trisomy 21 ◽  
Gene Dosage ◽  
Inducible Promoters ◽  
C Elegans ◽  
Dual Specificity ◽  
Specificity Protein

Abstract The pathology of trisomy 21/Down syndrome includes cognitive and memory deficits. Increased expression of the dual-specificity protein kinase DYRK1A kinase (DYRK1A) appears to play a significant role in the neuropathology of Down syndrome. To shed light on the cellular role of DYRK1A and related genes we identified three DYRK/minibrain-like genes in the genome sequence of Caenorhabditis elegans, termed mbk-1, mbk-2, and hpk-1. We found these genes to be widely expressed and to localize to distinct subcellular compartments. We isolated deletion alleles in all three genes and show that loss of mbk-1, the gene most closely related to DYRK1A, causes no obvious defects, while another gene, mbk-2, is essential for viability. The overexpression of DYRK1A in Down syndrome led us to examine the effects of overexpression of its C. elegans ortholog mbk-1. We found that animals containing additional copies of the mbk-1 gene display behavioral defects in chemotaxis toward volatile chemoattractants and that the extent of these defects correlates with mbk-1 gene dosage. Using tissue-specific and inducible promoters, we show that additional copies of mbk-1 can impair olfaction cell-autonomously in mature, fully differentiated neurons and that this impairment is reversible. Our results suggest that increased gene dosage of human DYRK1A in trisomy 21 may disrupt the function of fully differentiated neurons and that this disruption is reversible.

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.

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