scholarly journals Loss of SEC-23 in Caenorhabditis elegans Causes Defects in Oogenesis, Morphogenesis, and Extracellular Matrix Secretion

2003 ◽  
Vol 14 (11) ◽  
pp. 4414-4426 ◽  
Author(s):  
Brett Roberts ◽  
Caroline Clucas ◽  
Iain L. Johnstone
Keyword(s):  
Caenorhabditis Elegans ◽  
Rapid Onset ◽  
Transport Pathway ◽  
Early Maturation ◽  
C Elegans ◽  
Larval Stages ◽  
Membrane Surfaces ◽  
Golgi Transport ◽  
Adult Hermaphrodite ◽  
Extracellular Matrix Secretion

SEC-23 is a component of coat protein complex II (COPII)-coated vesicles involved in the endoplasmic reticulum-to-Golgi transport pathway of eukaryotes. During postembryonic life, Caenorhabditis elegans is surrounded by a collagenous exoskeleton termed the cuticle. From a screen for mutants defective in cuticle secretion, we identified and characterized a sec-23 mutant of C. elegans. By sequence homology, C. elegans has only the single sec-23 gene described herein. In addition to the cuticle secretion defect, mutants fail to complete embryonic morphogenesis. However, they progress through the earlier stages of embryogenesis, including gastrulation, and achieve substantial morphogenesis before death. We demonstrated a maternal component of SEC-23 function sufficient for progression through the earlier stages of embryogenesis and explaining the limited phenotype of the zygotic mutant. By RNA-mediated interference, we investigated the effects of perturbing COPII function during various postembryonic stages. During larval stages, major defects in cuticle synthesis and molting were observed. In the adult hermaphrodite, reduction of SEC-23 function by RNA-mediated interference caused a rapid onset of sterility, with defects in oogenesis including early maturation of the germline nuclei, probably a result of the observed loss of the GLP-1 receptor from the membrane surfaces adjacent to the developing germline nuclei.

scholarly journals Mutator Foci Are Regulated by Developmental Stage, RNA, and the Germline Cell Cycle in Caenorhabditis elegans

2020 ◽  
Vol 10 (10) ◽  
pp. 3719-3728 ◽  
Author(s):  
Celja J. Uebel ◽  
Dana Agbede ◽  
Dylan C. Wallis ◽  
Carolyn M. Phillips
Keyword(s):  
Cell Cycle ◽  
Caenorhabditis Elegans ◽  
Epigenetic Inheritance ◽  
Germline Cell ◽  
Late Pachytene ◽  
Larval Stages ◽  
Transcriptional Inhibition ◽  
Male Germline ◽  
Early Embryos ◽  
Adult Hermaphrodite

RNA interference is a crucial gene regulatory mechanism in Caenorhabditis elegans. Phase-separated perinuclear germline compartments called Mutator foci are a key element of RNAi, ensuring robust gene silencing and transgenerational epigenetic inheritance. Despite their importance, Mutator foci regulation is not well understood, and observations of Mutator foci have been largely limited to adult hermaphrodite germlines. Here we reveal that punctate Mutator foci arise in the progenitor germ cells of early embryos and persist throughout all larval stages. They are additionally present throughout the male germline and in the cytoplasm of post-meiotic spermatids, suggestive of a role in paternal epigenetic inheritance. In the adult germline, transcriptional inhibition results in a pachytene-specific loss of Mutator foci, indicating that Mutator foci are partially reliant on RNA for their stability. Finally, we demonstrate that Mutator foci intensity is modulated by the stage of the germline cell cycle and specifically, that Mutator foci are brightest and most robust in the mitotic cells, transition zone, and late pachytene of adult germlines. Thus, our data defines several new factors that modulate Mutator foci morphology which may ultimately have implications for efficacy of RNAi in certain cell stages or environments.

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.

Melting dsDNA donor molecules potentiates precision genome editing in C. elegans

2020 ◽  
Author(s):  
Krishna S. Ghanta ◽  
Craig C. Mello
Keyword(s):  
Caenorhabditis Elegans ◽  
Genome Editing ◽  
Germ Cells ◽  
Multiple Injections ◽  
C Elegans ◽  
Homology Directed Repair ◽  
Selection Strategies ◽  
Double Stranded Dna ◽  
Adult Hermaphrodite

ABSTRACTCRISPR genome editing has revolutionized genetics in many organisms. In the nematode Caenorhabditis elegans one injection into each of the two gonad arms of an adult hermaphrodite exposes hundreds of meiotic germ cells to editing mixtures, permitting the recovery of multiple indels or small precision edits from each successfully injected animal. Unfortunately, particularly for long insertions, editing efficiencies can vary widely, necessitating multiple injections, and often requiring co-selection strategies. Here we show that melting double stranded DNA (dsDNA) donor molecules prior to injection increases the frequency of precise homology-directed repair (HDR) by several fold for longer edits. We describe troubleshooting strategies that enable consistently high editing efficiencies resulting, for example, in up to 100 independent GFP knock-ins from a single injected animal. These efficiencies make C. elegans by far the easiest metazoan to genome edit, removing barriers to the use and adoption of this facile system as a model for understanding animal biology.

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.

Analysis of the VPE sequences in the Caenorhabditis elegans vit-2 promoter with extrachromosomal tandem array-containing transgenic strains

1994 ◽  
Vol 14 (1) ◽  
pp. 484-491
Author(s):  
M MacMorris ◽  
J Spieth ◽  
C Madej ◽  
K Lea ◽  
T Blumenthal
Keyword(s):  
Caenorhabditis Elegans ◽  
Fusion Gene ◽  
Caenorhabditis Briggsae ◽  
Unique Role ◽  
Promoter Function ◽  
C Elegans ◽  
Low Copy Number ◽  
Genes Encoding ◽  
Transgenic Lines ◽  
Adult Hermaphrodite

The Caenorhabditis elegans vit genes, encoding vitellogenins, are abundantly expressed in the adult hermaphrodite intestine. Two repeated elements, vit promoter element 1 (VPE1 [TGTCAAT]) and VPE2 (CTGATAA), have been identified in the 5' flanking DNA of each of the vit genes of C. elegans and Caenorhabditis briggsae. These elements have previously been shown to be needed for correctly regulated expression of a vit-2/vit-6 fusion gene in low-copy-number, integrated transgenes. Here we extend the analysis of the function of VPE1 and VPE2 by using transgenic lines carrying large, extrachromosomal arrays of the test genes. The results validate the use of such arrays for transgenic analysis of gene regulation in C. elegans, by confirming previous findings showing that the VPE1 at -45 and both VPE2s are sites of activation. Additional experiments now indicate that when the -45 VPE1 is inverted or replaced by a VPE2, nearly total loss of promoter function results, suggesting that the highly conserved -45 VPE1 plays a unique role in vit-2 promoter function. In contrast, single mutations eliminating the three upstream VPE1s are without effect. However, in combination in double and triple mutants, these upstream VPE1 mutations cause drastic reductions in expression levels. The -150 VPE2 can be replaced by a XhoI site (CTCGAG), and the -90 VPE2 can be eliminated, as long as the overlapping VPE1 is left intact, but when these two replacements are combined, activity is lost. Thus, the promoter must have at least one VPE2 and it must have at least two VPE1s, one at -45 and one additional upstream element.

scholarly journals The Emergent Connectome in Caenorhabditis elegans Embryogenesis

2017 ◽  
Author(s):  
◽  
Bradly Alicea
Keyword(s):  
Nervous System ◽  
Caenorhabditis Elegans ◽  
Cell Lineage ◽  
Model Organism ◽  
Secondary Data ◽  
Building Blocks ◽  
C Elegans ◽  
Differentiated Cells ◽  
Adult Hermaphrodite ◽  
Nervous System Function

ABSTRACTThe relatively new field of connectomics provides us with a unique window into nervous system function. In the model organism Caenorhabditis elegans, this promise is even greater due to the relatively small number of cells (302) in its nervous system. While the adult C. elegans connectome has been characterized, the emergence of these networks in development has yet to be established. In this paper, we approach this problem using secondary data describing the birth times of terminally-differentiated cells as they appear in the embryo and a connectomics model for nervous system cells in the adult hermaphrodite. By combining these two sources of data, we can better understand patterns that emerge in an incipient connectome. This includes identifying at what point in embryogenesis the cells of a connectome first comes into being, potentially observing some of the earliest neuron-neuron interactions, and making comparisons between the formally-defined connectome and developmental cell lineages. An analysis is also conducted to root terminally-differentiated cells in their developmental cell lineage precursors. This reveals subnetworks with different properties at 300 minutes of embryogenesis. Additional investigations reveal the spatial position of neuronal cells born during pre-hatch development, both within and outside the connectome model, in the context of all developmental cells in the embryo. Overall, these analyses reveal important information about the birth order of specific cells in the connectome, key building blocks of global connectivity, and how these structures correspond to key events in early development.

scholarly journals Mutator foci are regulated by developmental stage, RNA, and the germline cell cycle in Caenorhabditis elegans

2020 ◽  
Author(s):  
Celja J. Uebel ◽  
Dana Agbede ◽  
Dylan C. Wallis ◽  
Carolyn M. Phillips
Keyword(s):  
Cell Cycle ◽  
Caenorhabditis Elegans ◽  
Epigenetic Inheritance ◽  
Germline Cell ◽  
Late Pachytene ◽  
Larval Stages ◽  
Transcriptional Inhibition ◽  
Male Germline ◽  
Early Embryos ◽  
Adult Hermaphrodite

ABSTRACTRNA interference is a crucial gene regulatory mechanism in Caenorhabditis elegans. Phase-separated perinuclear germline compartments called Mutator foci are a key element of RNAi, ensuring robust gene silencing and transgenerational epigenetic inheritance. Despite their importance, Mutator foci regulation is not well understood, and observations of Mutator foci have been largely limited to adult hermaphrodite germlines. Here we reveal that punctate Mutator foci arise in the progenitor germ cells of early embryos and persist throughout all larval stages. They are additionally present throughout the male germline and in the cytoplasm of post-meiotic spermatids, suggestive of a role in paternal epigenetic inheritance. In the adult germline, transcriptional inhibition results in a pachytene-specific loss of Mutator foci, indicating that Mutator foci are partially reliant on RNA for their stability. Finally, we demonstrate that Mutator foci intensity is modulated by the stage of the germline cell cycle and specifically, that Mutator foci are brightest and most robust in the mitotic cells, transition zone, and late pachytene of adult germlines. Thus, our data defines several new factors that modulate Mutator foci morphology which may ultimately have implications for efficacy of RNAi in certain cell stages or environments.

scholarly journals Analysis of the VPE sequences in the Caenorhabditis elegans vit-2 promoter with extrachromosomal tandem array-containing transgenic strains.

1994 ◽  
Vol 14 (1) ◽  
pp. 484-491 ◽  
Author(s):  
M MacMorris ◽  
J Spieth ◽  
C Madej ◽  
K Lea ◽  
T Blumenthal
Keyword(s):  
Caenorhabditis Elegans ◽  
Fusion Gene ◽  
Caenorhabditis Briggsae ◽  
Unique Role ◽  
Promoter Function ◽  
C Elegans ◽  
Low Copy Number ◽  
Genes Encoding ◽  
Transgenic Lines ◽  
Adult Hermaphrodite

The Caenorhabditis elegans vit genes, encoding vitellogenins, are abundantly expressed in the adult hermaphrodite intestine. Two repeated elements, vit promoter element 1 (VPE1 [TGTCAAT]) and VPE2 (CTGATAA), have been identified in the 5' flanking DNA of each of the vit genes of C. elegans and Caenorhabditis briggsae. These elements have previously been shown to be needed for correctly regulated expression of a vit-2/vit-6 fusion gene in low-copy-number, integrated transgenes. Here we extend the analysis of the function of VPE1 and VPE2 by using transgenic lines carrying large, extrachromosomal arrays of the test genes. The results validate the use of such arrays for transgenic analysis of gene regulation in C. elegans, by confirming previous findings showing that the VPE1 at -45 and both VPE2s are sites of activation. Additional experiments now indicate that when the -45 VPE1 is inverted or replaced by a VPE2, nearly total loss of promoter function results, suggesting that the highly conserved -45 VPE1 plays a unique role in vit-2 promoter function. In contrast, single mutations eliminating the three upstream VPE1s are without effect. However, in combination in double and triple mutants, these upstream VPE1 mutations cause drastic reductions in expression levels. The -150 VPE2 can be replaced by a XhoI site (CTCGAG), and the -90 VPE2 can be eliminated, as long as the overlapping VPE1 is left intact, but when these two replacements are combined, activity is lost. Thus, the promoter must have at least one VPE2 and it must have at least two VPE1s, one at -45 and one additional upstream element.

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.

scholarly journals Melting dsDNA Donor Molecules Greatly Improves Precision Genome Editing in Caenorhabditis elegans

Genetics ◽  
2020 ◽  
Vol 216 (3) ◽  
pp. 643-650
Author(s):  
Krishna S. Ghanta ◽  
Craig C. Mello
Keyword(s):  
Caenorhabditis Elegans ◽  
Genome Editing ◽  
Germ Cells ◽  
Multiple Injections ◽  
C Elegans ◽  
Homology Directed Repair ◽  
Double Stranded Dna ◽  
Adult Hermaphrodite

CRISPR genome editing has revolutionized genetics in many organisms. In the nematode Caenorhabditis elegans, one injection into each of the two gonad arms of an adult hermaphrodite exposes hundreds of meiotic germ cells to editing mixtures, permitting the recovery of multiple indels or small precision edits from each successfully injected animal. Unfortunately, particularly for long insertions, editing efficiencies can vary widely, necessitating multiple injections, and often requiring coselection strategies. Here, we show that melting double-stranded DNA (dsDNA) donor molecules prior to injection increases the frequency of precise homology-directed repair (HDR) by several fold for longer edits. We describe troubleshooting strategies that enable consistently high editing efficiencies resulting, for example, in up to 100 independent GFP knock-ins from a single injected animal. These efficiencies make C. elegans by far the easiest metazoan to genome edit, removing barriers to the use and adoption of this facile system as a model for understanding animal biology.

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