scholarly journals Microfluidic-based imaging of complete C. elegans larval development

Development ◽  
2021 ◽  
Author(s):  
Simon Berger ◽  
Silvan Spiri ◽  
Andrew deMello ◽  
Alex Hajnal
Keyword(s):  
Imaging Method ◽  
Cover Glass ◽  
Vulval Development ◽  
Minimal Impact ◽  
C Elegans ◽  
Larval Stages ◽  
Stable Orientation ◽  
On Chip ◽  
First Time ◽  
Hydraulic Valve

Several microfluidic-based methods for C. elegans imaging have recently been introduced. Existing methods either permit imaging across multiple larval stages without maintaining a stable worm orientation, or allow for very good immobilization but are only suitable for shorter experiments. Here, we present a novel microfluidic imaging method, which allows parallel live-imaging across multiple larval stages, while maintaining worm orientation and identity over time. This is achieved through an array of microfluidic trap channels carefully tuned to maintain worms in a stable orientation, while allowing growth and molting to occur. Immobilization is supported by an active hydraulic valve, which presses worms onto the cover glass during image acquisition only. In this way, excellent quality images can be acquired with minimal impact on worm viability or developmental timing. The capabilities of the devices are demonstrated by observing the hypodermal seam and P cell divisions and, for the first time, the entire process of vulval development from induction to the end of morphogenesis. Moreover, we demonstrate feasibility of on-chip RNAi by perturbing basement membrane breaching during anchor cell invasion.

scholarly journals Microfluidic-based imaging of complete C. elegans larval development

2021 ◽  
Author(s):  
Simon Berger ◽  
Silvan Spiri ◽  
Andrew deMello ◽  
Alex Hajnal
Keyword(s):  
Imaging Method ◽  
Cover Glass ◽  
Vulval Development ◽  
C Elegans ◽  
Larval Stages ◽  
Seam Cell ◽  
Time Observation ◽  
On Chip ◽  
First Time

Several microfluidic-based methods for long-term C. elegans imaging have been introduced in recent years, allowing real-time observation of previously inaccessible processes. The ex-isting methods either permit imaging across multiple larval stages without maintaining a stable worm orientation, or allow for very good immobilization but are only suitable for shorter experiments. Here, we present a novel microfluidic imaging method, which allows parallel live-imaging across multiple larval stages, while delivering excellent immobilization and maintaining worm orientation and identity over time. This is achieved by employing an array of microfluidic trap channels carefully tuned to maintain worms in a stable orienta-tion, while allowing growth and molting to occur. Immobilization is supported by an active hydraulic valve, which presses worms onto the cover glass during image acquisition, with the animals remaining free for most of an experiment. Excellent quality images can be ac-quired of multiple worms in parallel, with little impact of the imaging method on worm via-bility or developmental timing. The capabilities of this methodology are demonstrated by observing the hypodermal seam cell divisions and, for the first time, the entire process of vulval development from induction to the end of morphogenesis. Moreover, we demonstrate RNAi on-chip, which allows for perturbation of dynamic developmental processes, such as basement membrane breaching during anchor cell invasion.

Microbubble Array as a Versatile Tool for On-Chip Worm Processing

2012 ◽  
Author(s):  
Gan Yu ◽  
Ali Hashmi ◽  
Yuhao Xu ◽  
Hyuck-Jin Kwon ◽  
Xiaolin Chen ◽  
...  
Keyword(s):  
Microfluidic Device ◽  
Trapping Efficiency ◽  
Flow Conditions ◽  
C Elegans ◽  
Novel Technique ◽  
Versatile Tool ◽  
On Chip ◽  
First Time

In this study, we present a novel technique for achieving precise trapping, enrichment and manipulation of C. elegans using an array of oscillating microbubbles in a simple microfluidic device. We demonstrate a high trapping efficiency and enrichment of C. elegans characterized by the flow conditions. We also demonstrate for the first time an on-chip manipulation of the pathways of a single C. elegans through temporal actuation of bubbles.

scholarly journals Functional Characterization of the Adenylyl Cyclase Genesgs-1by Analysis of a Mutational Spectrum inCaenorhabditis elegans

Genetics ◽  
2002 ◽  
Vol 161 (1) ◽  
pp. 133-142 ◽  
Author(s):  
Celine Moorman ◽  
Ronald H A Plasterk
Keyword(s):  
Life Span ◽  
Adenylyl Cyclase ◽  
Neuronal Degeneration ◽  
Functional Characterization ◽  
Adenylyl Cyclases ◽  
Loss Of Function ◽  
C Elegans ◽  
Larval Stages ◽  
Pharyngeal Pumping

AbstractThe sgs-1 (suppressor of activated Gαs) gene encodes one of the four adenylyl cyclases in the nematode C. elegans and is most similar to mammalian adenylyl cyclase type IX. We isolated a complete loss-of-function mutation in sgs-1 and found it to result in animals with retarded development that arrest in variable larval stages. sgs-1 mutant animals exhibit lethargic movement and pharyngeal pumping and (while not reaching adulthood) have a mean life span that is >50% extended compared to wild type. An extensive set of reduction-of-function mutations in sgs-1 was isolated in a screen for suppressors of a neuronal degeneration phenotype induced by the expression of a constitutively active version of the heterotrimeric Gαs subunit of C. elegans. Although most of these mutations change conserved residues within the catalytic domains of sgs-1, mutations in the less-conserved transmembrane domains are also found. The sgs-1 reduction-of-function mutants are viable and have reduced locomotion rates, but do not show defects in pharyngeal pumping or life span.

scholarly journals Associations of Phoretic Mites on Bark Beetles of the Genus Ips in the Black Sea Mountains of Turkey

Forests ◽  
2021 ◽  
Vol 12 (5) ◽  
pp. 516
Author(s):  
Cihan Cilbircioğlu ◽  
Marta Kovač ◽  
Milan Pernek
Keyword(s):  
Black Sea ◽  
Bark Beetles ◽  
Pinus Nigra ◽  
Mite Species ◽  
Tree Bark ◽  
The Black Sea ◽  
Forest Pests ◽  
Larval Stages ◽  
Suitable Habitats ◽  
First Time

Phoretic mites use bark beetles for transportation to new, suitable habitats. Some phoretic mites act as predators and parasitoids of the bark beetles’ immature stages, especially egg and early larval stages, and are potential agents for the biological control of scolytine forest pests. Mites live very frequently in relationships with other invertebrates. Many are found in association with various species of bark beetles. Here, a total of 41 specimens of different bark beetles of the genus Ips (Ips acuminatus, Ips sexdentatus and Ips typographus) were studied for presence, species composition, and abundance of phoretic mites. The beetles were collected on dead wood and parts of tree bark of Pinus nigra, Pinus sylvestris and Picea abies in the Black Sea Mountains in Kastamonu and Artin Province of Turkey. A total of nine mite species were found, including Dendrolaelaps quadrisetus, Ereynetes sp., Histiostoma piceae, Paraleius cf. leontonychus, Pleuronectocelaeno barbara., Proctolaelaps hystricoides, Schizostethus simulatrix, Trichouropoda lamellosa and Uroobovellaipidis. All species are identified for the first time within Turkish fauna.

hlh-12, a gene that is necessary and sufficient to promote migration of gonadal regulatory cells in C. elegans, evolved within the Caenorhabditis clade

Genetics ◽  
2021 ◽  
Author(s):  
Hana E Littleford ◽  
Karin Kiontke ◽  
David H A Fitch ◽  
Iva Greenwald
Keyword(s):  
Ectopic Expression ◽  
Morphological Diversity ◽  
Nematode Species ◽  
Bhlh Protein ◽  
Vulval Development ◽  
C Elegans ◽  
Form And Function ◽  
Somatic Gonad ◽  
And Function ◽  
Necessary And Sufficient

Abstract Specialized cells of the somatic gonad primordium of nematodes play important roles in the final form and function of the mature gonad. C. elegans hermaphrodites are somatic females that have a two-armed, U-shaped gonad that connects to the vulva at the midbody. The outgrowth of each gonad arm from the somatic gonad primordium is led by two female Distal Tip Cells (fDTC), while the Anchor Cell (AC) remains stationary and central to coordinate uterine and vulval development. The bHLH protein HLH-2 and its dimerization partners LIN-32 and HLH-12 had previously been shown to be required for fDTC specification. Here, we show that ectopic expression of both HLH-12 and LIN-32 in cells with AC potential transiently transforms them into fDTC-like cells. Furthermore, hlh-12 was known to be required for the fDTCs to sustain gonad arm outgrowth. Here, we show that ectopic expression of HLH-12 in the normally stationary AC causes displacement from its normal position, and that displacement likely results from activation of the leader program of fDTCs because it requires genes necessary for gonad arm outgrowth. Thus, HLH-12 is both necessary and sufficient to promote gonadal regulatory cell migration. As differences in female gonadal morphology of different nematode species reflect differences in the fate or migratory properties of the fDTCs or of the AC, we hypothesized that evolutionary changes in the expression of hlh-12 may underlie evolution of such morphological diversity. However, we were unable to identify an hlh-12 ortholog outside of Caenorhabditis. Instead, by performing a comprehensive phylogenetic analysis of all Class II bHLH proteins in multiple nematode species, we found that HLH-12 evolved within the Caenorhabditis clade, possibly by duplicative transposition of hlh-10. Our analysis suggests that control of gene regulatory hierarchies for gonadogenesis can be remarkably plastic during evolution without adverse phenotypic consequence.

scholarly journals The retromer complex regulates C. elegans development and mammalian ciliogenesis

2021 ◽  
Author(s):  
Shuwei Xie ◽  
Ellie Smith ◽  
Carter Dierlam ◽  
Danita Mathew ◽  
Angelina Davis ◽  
...  
Keyword(s):  
Potential Function ◽  
Primary Cilium ◽  
Mammalian Cells ◽  
Primary Cilia ◽  
Vulval Development ◽  
Sorting Nexin ◽  
Capping Protein ◽  
C Elegans ◽  
Retromer Complex ◽  
Mother Centriole

The mammalian retromer is comprised of subunits VPS26, VPS29 and VPS35, and a more loosely-associated sorting nexin (SNX) heterodimer. Despite known roles for the retromer in multiple trafficking events in yeast and mammalian cells, its role in development is poorly understood, and its potential function in primary ciliogenesis remains unknown. Using CRISPR-Cas9 editing, we demonstrated that vps-26 homozygous knockout C. elegans have reduced brood sizes and impaired vulval development, as well as decreased body length which has been linked to defects in primary ciliogenesis. Since many endocytic proteins are implicated in the generation of primary cilia, we addressed whether the retromer regulates ciliogenesis in mammalian cells. We observed VPS35 localized to the primary cilium, and depletion of VPS26, VPS35 or SNX1/SNX5 led to decreased ciliogenesis. Retromer also coimmunoprecipitated with the capping protein, CP110, and was required for its removal from the mother centriole. Herein, we characterize new roles for the retromer in C. elegans development and in the regulation of ciliogenesis in mammalian cells, and suggest a novel role for the retromer in CP110 removal from the mother centriole.

Sign in / Sign up

Export Citation Format

Share Document