This work was supported by grants from the Natural Sciences and Engineering Research Council (NSERC) of Canada and The Canada Foundation for Innovation. Thanks to Dr Paul Mains (University of Calgary, Calgary, Canada) for let-502(sb118ts) strain. Some of the strains were provided by the Caenorhabditis Genetics Center, which is funded by NIH Office of Research Infrastructure Programs (P40 OD010440).

<ol>
	<li>Corsi, A. K., Wightman, B., Chalfie, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Transparent+Window+into+Biology:+A+Primer+on+Caenorhabditis+elegans.">A Transparent Window into Biology: A Primer on Caenorhabditis elegans.</a> Genetics. 200, 387-407 (2015).</li><li>Priess, J. R., Hirsh, D. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Caenorhabditis+elegans+morphogenesis:+the+role+of+the+cytoskeleton+in+elongation+of+the+embryo.">Caenorhabditis elegans morphogenesis: the role of the cytoskeleton in elongation of the embryo.</a> Developmental biology. 117, 156-173 (1986).</li><li>Zhang, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+tension-induced+mechanotransduction+pathway+promotes+epithelial+morphogenesis.">A tension-induced mechanotransduction pathway promotes epithelial morphogenesis.</a> Nature. 471, 99-103 (2011).</li><li>Diogon, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+RhoGAP+RGA-2+and+LET-502/ROCK+achieve+a+balance+of+actomyosin-dependent+forces+in+C.+elegans+epidermis+to+control+morphogenesis.">The RhoGAP RGA-2 and LET-502/ROCK achieve a balance of actomyosin-dependent forces in C. elegans epidermis to control morphogenesis.</a> Development. 134, 2469-2479 (2007).</li><li>Gally, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Myosin+II+regulation+during+C.+elegans+embryonic+elongation:+LET-502/ROCK,+MRCK-1+and+PAK-1,+three+kinases+with+different+roles.">Myosin II regulation during C. elegans embryonic elongation: LET-502/ROCK, MRCK-1 and PAK-1, three kinases with different roles.</a> Development. 136, 3109-3119 (2009).</li><li>Piekny, A. J., Wissmann, A., Mains, P. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Embryonic+morphogenesis+in+Caenorhabditis+elegans+integrates+the+activity+of+LET-502+Rho-binding+kinase,+MEL-11+myosin+phosphatase,+DAF-2+insulin+receptor+and+FEM-2+PP2c+phosphatase.">Embryonic morphogenesis in Caenorhabditis elegans integrates the activity of LET-502 Rho-binding kinase, MEL-11 myosin phosphatase, DAF-2 insulin receptor and FEM-2 PP2c phosphatase.</a> Genetics. 156, 1671-1689 (2000).</li><li>Martin, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=pix-1+controls+early+elongation+in+parallel+with+mel-11+and+let-502+in+Caenorhabditis+elegans.">pix-1 controls early elongation in parallel with mel-11 and let-502 in Caenorhabditis elegans.</a> PloS one. 9, e94684 (2014).</li><li>Sulston, J., Hodgkin, J., Wood, W. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Methods. , 587-606 (1988).</li><li>Andersen, E. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Powerful+New+Quantitative+Genetics+Platform,+Combining+Caenorhabditis+elegans+High-Throughput+Fitness+Assays+with+a+Large+Collection+of+Recombinant+Strains.">A Powerful New Quantitative Genetics Platform, Combining Caenorhabditis elegans High-Throughput Fitness Assays with a Large Collection of Recombinant Strains.</a> G3 (Bethesda). 5, 911-920 (2015).</li><li>Boulier, E. L., Jenna, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+dissection+of+Caenorhabditis+elegans+embryogenesis+using+RNA+interference+and+flow+cytometry.">Genetic dissection of Caenorhabditis elegans embryogenesis using RNA interference and flow cytometry.</a> Methods Mol Biol. 550, 181-194 (2009).</li><li>Verster, A. J., Ramani, A. K., McKay, S. J., Fraser, A. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparative+RNAi+screens+in+C.+elegans+and+C.+briggsae+reveal+the+impact+of+developmental+system+drift+on+gene+function.">Comparative RNAi screens in C. elegans and C. briggsae reveal the impact of developmental system drift on gene function.</a> PLoS genetics. 10, e1004077 (2014).</li><li>Ramani, A. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+majority+of+animal+genes+are+required+for+wild-type+fitness.">The majority of animal genes are required for wild-type fitness.</a> Cell. 148, 792-802 (2012).</li><li>So, S., Miyahara, K., Ohshima, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Control+of+body+size+in+C.+elegans+dependent+on+food+and+insulin/IGF-1+signal.">Control of body size in C. elegans dependent on food and insulin/IGF-1 signal.</a> Genes to cells : devoted to molecular &amp; cellular mechanisms. 16, 639-651 (2011).</li><li>Dineen, A., Gaudet, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TGF-beta+signaling+can+act+from+multiple+tissues+to+regulate+C.+elegans+body+size.">TGF-beta signaling can act from multiple tissues to regulate C. elegans body size.</a> BMC developmental biology. 14, 43 (2014).</li><li>Tan, L., Wang, S., Wang, Y., He, M., Liu, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bisphenol+A+exposure+accelerated+the+aging+process+in+the+nematode+Caenorhabditis+elegans.">Bisphenol A exposure accelerated the aging process in the nematode Caenorhabditis elegans.</a> Toxicology letters. 235, 75-83 (2015).</li><li>Yu, Z., Yin, D., Deng, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+combinational+effects+between+sulfonamides+and+metals+on+nematode+Caenorhabditis+elegans.">The combinational effects between sulfonamides and metals on nematode Caenorhabditis elegans.</a> Ecotoxicology and environmental safety. 111, 66-71 (2015).</li></ol>

The authors declare that they have no competing financial interests.

Dieses Protokoll beschreibt neue Metriken frühen und späten Phasen der embryonalen Dehnung zu charakterisieren. Im Bereich 1 ist der kritische Schritt die mögliche Anwesenheit von Bakterien auf dem Pad. Die Embryonen werden hermetisch zwischen dem Kissen und dem Deckglas während der Bilderfassung eingeschlossen ist. die Folie Abdichtung ist erforderlich während der Erfassung Desikkation der Tiere zu vermeiden, dass mehr als zwei Stunden dauert. Unseres Wissens nach keiner der Versiegelu...

Seit fast 50 Jahren gründeten die Nematode Caenorhabditis elegans sich als leistungsfähiges Modell wichtige Fragen in der Entwicklung, der Neurobiologie, evolution, Wirt-Pathogen - Interaktionen usw. 1 Die Stärke dieses Modells in der Studie der Entwicklung zu studieren , liegt in: seiner kurzen Lebenszyklus von 3 Tagen; die Leichtigkeit, mit der diese Tiere gentechnisch verändert werden; seine Transparenz, die die Beobachtung der Zell Verschiebung und Morphologie in lebenden Tieren und deren Entwicklung, die vor allem Extrauterin- ist ermöglicht. Die Entwicklungsstadien des Nematoden umfassen Embryonalentwicklung und vier Larvenstadien (L1 bis L4), gefolgt von Erwachsenenalter. Während der embryonalen Entwicklung, zog epidermal Morphogenese beträchtliche Aufmerksamkeit auf seine Fähigkeit, um ein besseres Verständnis davon, wie Epithelzellen wandern als Gruppe zu ermöglichen, wie sie ihre junctions reorganisieren und ändern ihre individuellen Morphologie sowie deren relative Positionierung innerhalb eines funktionellen Epithels.Epidermal Morphogenese ist in vier Stufen unterteilt: dorsale Einlagerungs bestehend in der Reorganisation der dorsalen epidermalen Zellen, als der Unterhaut bezeichnet; ventralen Gehäuse, in der Migration von ventralen hypodermal Zellen in Richtung der ventralen Mittellinie aus so den Embryo in einer epithelialen Zellschicht umhüllt; Früh- und Spät Dehnung Umwandlung der bohnenförmigen Embryos in vermiform Larven. Im Anschluss an die Morphogenese, Embryo Luke und L1-Larven beginnen mit den verfügbaren Bakterien in ihrer unmittelbaren Umgebung ernähren. Embryonic Dehnung ist daher eine späte Phase der Embryonalentwicklung. Es besteht aus der Verlängerung des Embryos entlang seiner Längsachse und eine Verringerung seines Querdurchmesser. Dies bedeutet eine drastische Änderung der Form der hypodermalen Zellen. Dehnung ist unterteilt in eine frühe und eine späte Phase. Die frühe Phase beginnt mit dem Komma Bühne und endet , wenn die Körperwand Muskeln in w bei 1,75-facher Etappenvertragsild Typ (wt) Embryonen - entsprechend Embryonen , die 1,75-fache in der Länge im Vergleich zu nicht verlängert Embryonen. Morphogene Prozesse in diesem Stadium auftreten , werden in erster Linie durch die Kontraktion von fädigen Aktinbündel (FB) am apikalen Pol hypodermal Zellen angeordnet angetrieben, die ihre Dehnung entlang der anteroposterioren Achse des Embryos 2 fahren. Die Kontraktion der FBs ist die Kontrolle durch die Phosphorylierung von Myosin-Leichtketten durch drei Kinasen LET-502 / ROCK, MRCK-1 und PAK-1 5. Die Spätphase der Verlängerung beginnt, wenn die Körperwand Muskeln funktionsfähig werden und Contracting beginnen. Es geht um Mechanotransduktion Signalisierung von den Körperwandmuskulatur zu den dorsalen und ventralen hypodermal Zellen und endet , wenn die Tiere 3 schlüpfen. Dehnungsfehler werden durch den Prozentsatz der Tiere als Embryonen (Embryonale Letalität; Emb) sterben im Allgemeinen gekennzeichnet und diejenigen zu verhaften ihre Entwicklung als L1-Larven (Larven Verhaftung Phänotyp; Lva) aufweist und deutlich kürzer als Gew. Bezeichnung des Stadium der Entwicklungsstopp erfordert mikroskopische Beobachtung von toten Embryonen und verhaftet Larvae 3-6. Es wurde vor kurzem gezeigt , dass mehrere Gene, wie die Cdc42 / Rac Regler und Effektor pix-1 und pak-1, während morphogenic Prozesse steuern sowohl frühe als auch späte Dehnung 3,7. Wir zeigten auch vor kurzem , dass morphogenic Prozesse entlang der anteroposterioren Achse der Embryonen während der frühen Verlängerung 3 7 unterscheiden. Diese Erkenntnisse die Entwicklung neuer Metriken motiviert speziell frühe oder späte Verlängerung Stadien und anderen Metriken Targeting ermöglicht die Charakterisierung der Morphologie der Embryonen entlang ihrer anteroposterioren Achse während der frühen Dehnung. Diese neuen Verfahren bestehen, um die Länge von Embryonen zu Beginn und am Ende des frühen Dehnung sowie die Breite der bei der Messung erAnzeigen und Schwänzen. 7 Zwei Protokolle wurden auch die Länge der frisch geschlüpften Larven zu messen entwickelt, synchronisiert auf L1 Stufe 7. Die Eischalen der Embryonen schützen sie gegen alkalischen Hypochlorit-Behandlung während Larven, Erwachsene und Bakterien in den Kulturmedien durch die Behandlung gelöst sind. Diese Behandlung wird dann verwendet , Embryonen zu reinigen , aus einem nicht-synchronisierten Population eine Mehrheit von gut ernährt Erwachsene 8 enthält. Lebensmittel Einschränkung verwendet frisch geschlüpften Larven zu synchronisieren. Messen der Länge dieser Larven wird dann verwendet, Verlängerung Defekte zu detektieren. Diese Messung wird über die Messung der verhafteten Larven auf Kulturplatten bevorzugt, da Larven, die aus nicht vollständig länglichen Embryonen schlüpfen können &quot;normale Länge&quot; erholen, wenn Fütterung aber ihre reduzierte Größe beibehalten, wenn in Abwesenheit von Lebensmitteln festgenommen. Hier präsentieren wir detaillierte Protokolle ermöglichen die Messung der lenge von Embryonen sowie die Breite des Kopfes und der Schwanz mit Zeitraffer-DIC-Mikroskopie und Bildanalyse (Protokoll 1) verlängert wird. Wir bieten auch detaillierte Protokolle über die Länge der synchronisierten Larven mittels Bildanalyse (Protokoll 2) und Durchflusszytometrie (Protokoll 3) zu messen.

<table><tbody><tr><td>Agar&amp;nbsp;</td><td>BioShop</td><td>AGR001.500</td><td></td></tr><tr><td>Agarose</td><td>Bioshop</td><td>AGA001.500</td><td></td></tr><tr><td>CaCl&lt;sub&gt;2&lt;/sub&gt; (Calcium Chloride)</td><td>Bio Basic Inc.</td><td>CT1330</td><td></td></tr><tr><td>Cholesterol</td><td>Sigma-aldrich</td><td>C8667</td><td></td></tr><tr><td>Cleaning solution&amp;nbsp;</td><td>union Biometrica</td><td>300-5072-000</td><td></td></tr><tr><td>Glass coverslips</td><td>Fisherbrand</td><td>12-542B</td><td></td></tr><tr><td>Glass slides</td><td>Fisherbrand</td><td>12-552-3</td><td></td></tr><tr><td>High fluorescent control particles</td><td>union Biometrica</td><td>310-5071-001</td><td></td></tr><tr><td>K&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt; (potassium phosphate, dibasic)</td><td>Bio Basic Inc.</td><td>PB0447</td><td></td></tr><tr><td>K&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt; (potassium phosphate, monobasic)</td><td>Bio Basic Inc.</td><td>PB0445</td><td></td></tr><tr><td>MgSO&lt;sub&gt;4&lt;/sub&gt; (magnesium sulfate)</td><td>Sigma-aldrich</td><td>230391</td><td></td></tr><tr><td>Na&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt;( sodium phosphate, dibasic)</td><td>Bio Basic Inc.</td><td>SDB0487</td><td></td></tr><tr><td>NaCl (sodium chloride)</td><td>Bio Basic Inc.</td><td>DB0483</td><td></td></tr><tr><td>Pebeo Drawing Gum 45&amp;nbsp;ml</td><td>p&amp;eacute;b&amp;eacute;o</td><td>PDG033000</td><td>any art/craft store</td></tr><tr><td>Peptone</td><td>BioShop</td><td>PEP403.500</td><td></td></tr><tr><td>Sheath buffer</td><td>union Biometrica</td><td>300-5070-100</td><td></td></tr><tr><td>COPAS Biosort</td><td>union Biometrica</td><td></td><td></td></tr></tbody></table>

novel metrics to characterize embryonic elongation of the nematode caenorhabditis elegans

Dissecting the signaling pathways that control the alteration of morphogenic processes during embryonic development requires robust and sensitive metrics. Embryonic elongation of the nematode Caenorhabditis elegans is a late developmental stage consisting of the elongation of the embryo along its longitudinal axis. This developmental stage is controlled by intercellular communication between hypodermal cells and underlying body-wall muscles. These signaling mechanisms control the morphology of hypodermal cells by remodeling the cytoskeleton and the cell-cell junctions. Measurement of embryonic lethality and developmental arrest at larval stages as well as alteration of cytoskeleton and cell-cell adhesion structures in hypodermal and muscle cells are classical phenotypes that have been used for more than 25 years to dissect these signaling pathways. Recent studies required the development of novel metrics specifically targeting either early or late elongation and characterizing morphogenic defects along the antero-posterior axis of the embryo. Here, we provide detailed protocols enabling the accurate measurement of the length and the width of the elongating embryos as well as the length of synchronized larvae. These methods constitute useful tools to identify genes controlling elongation, to assess whether these genes control both early and late phases of this stage and are required evenly along the antero-posterior axis of the embryo.

Head-, Schwanz- und Kopf / Schwanz-Breite - Verhältnis sind Robust Metrics. Die Protokolle hier beschrieben wurden während der frühen Verlängerung 7. Pix-1 und pak-1 - Code jeweils für eine Guanin-Austauschfaktor Funktion der Regulatoren un...

Watch this Scientific Journal Video about Novel Metrics to Characterize Embryonic Elongation of the Nematode Caenorhabditis elegans at JoVE.com

Novel Metrics to Characterize Embryonic Elongation of the Nematode Caenorhabditis elegans

Novel Metrics zur Charakterisierung von Embryonic Dehnung des Nematode<em&gt; Caenorhabditis elegans</em

Here we detail protocols specifically designed to monitor morphogenic defects that occur during early and late phases of embryonic elongation of the nematode Caenorhabditis elegans. Ultimately, these protocols are designed to identify genes that regulate these phases and to characterize their differential requirements along the antero-posterior axis of the embryo.

Dissecting the signaling pathways that control the alteration of morphogenic processes during embryonic development requires robust and sensitive metrics. ...

novel-metrics-to-characterize-embryonic-elongation-nematode

Research

JoVE Journal

Neuroscience

Novel Metrics to Characterize Embryonic Elongation of the Nematode Caenorhabditis elegans

6.0K Aufrufe.  Université du Québec à Montréal. Here we detail protocols specifically designed to monitor morphogenic defects that occur during early and late phases of embryonic elongation of the nematode Caenorhabditis elegans. Ultimately, these protocols are designed to identify genes that regulate these phases and to characterize their differential requirements along the antero-posterior axis of the embryo. 

Serie: Novel Metrics to Characterize Embryonic Elongation of the Nematode Caenorhabditis elegans

C. elegans Tracking and Behavioral Measurement

We have developed instrumentation, image processing, and data analysis techniques to quantify the locomotory behavior of C. elegans as it crawls on the surface of an agar plate. For the study of the genetic, biochemical, and neuronal basis of behavior, C. elegans is an ideal organism because it is genetically tractable, amenable to microscopy, and shows a number of complex behaviors, including taxis, learning, and social interaction1,2. Behavioral analysis based on tracking the movements of worms as they crawl on agar plates have been particularly useful in the study of sensory behavior3, locomotion4, and general mutational phenotyping5. Our system works by moving the camera and illumination system as the worms crawls on a stationary agar plate, which ensures no mechanical stimulus is transmitted to the worm. Our tracking system is easy to use and includes a semi-automatic calibration feature. A challenge of all video tracking systems is that it generates an enormous amount of data that is intrinsically high dimensional. Our image processing and data analysis programs deal with this challenge by reducing the worms shape into a set of independent components, which comprehensively reconstruct the worms behavior as a function of only 3-4 dimensions6,7. As an example of the process we show that the worm enters and exits its reversal state in a phase specific manner.

C. elegans Tracking and Behavioral Measurement

We have developed a video-rate tracking microscope system that can record and quantify C. elegans behavior at high resolution and high speeds. We have also developed computational methods to reduce the dimensionality of the worm images to a fundamental set of measurements that completely describe the shape of the worm.

C. elegans Tracking und Behavioral Measurement

We have developed instrumentation, image processing, and data analysis techniques to quantify the locomotory behavior of C. elegans as it crawls on the ...

Forschung

Neurowissenschaften

Measuring Embryonic Viability and Brood Size in Caenorhabditis elegans

Caenorhabditis elegans is an excellent model organism for the study of meiosis, fertilization, and embryonic development. C. elegans exist as self-fertilizing hermaphrodites, which produce large broods of progeny-when males are present, they can produce even larger broods of cross progeny. Errors in meiosis, fertilization, and embryogenesis can be rapidly assessed as phenotypes of sterility, reduced fertility, or embryonic lethality. This article describes a method to determine embryonic viability and brood size in C. elegans. We demonstrate how to set up this assay by picking a worm onto an individual Modified Youngren's, Only Bacto-peptone (MYOB) plate, establish the appropriate timeframe to count viable progeny and non-viable embryos, and explain how to accurately count live worm specimens. This technique can be used to determine viability in self-fertilizing hermaphrodites as well as cross-fertilization by mating pairs. These relatively simple experiments are easily adoptable for new researchers, such as undergraduate students and first-year graduate students.

Measuring Embryonic Viability and Brood Size in Caenorhabditis elegans

Here, we present a general method to determine the embryonic viability and total number of embryos produced (brood) using the model organism C. elegans.

Messung der Embryonalviabilität und Brutgröße bei Caenorhabditis elegans

Caenorhabditis elegans is an excellent model organism for the study of meiosis, fertilization, and embryonic development. C. elegans exist as ...

Genetik

Isotropic Light-Sheet Microscopy and Automated Cell Lineage Analyses to Catalogue Caenorhabditis elegans Embryogenesis with Subcellular Resolution

Caenorhabditis elegans (C. elegans) stands out as the only organism in which the challenge of understanding the cellular origins of an entire nervous system can be observed, with single cell resolution, in vivo. Here, we present an integrated protocol for the examination of neurodevelopment in C. elegans embryos. Our protocol combines imaging, lineaging and neuroanatomical tracing of single cells in developing embryos. We achieve long-term, four-dimensional (4D) imaging of living C. elegans&#160;embryos with nearly isotropic spatial resolution through the use of Dual-view Inverted Selective Plane Illumination Microscopy (diSPIM). Nuclei and neuronal structures in the nematode embryos are imaged and isotropically fused to yield images with resolution of ~330 nm in all three dimensions. These minute-by-minute high-resolution 4D data sets are then analyzed to correlate definitive cell-lineage identities with gene expression and morphological dynamics at single-cell and subcellular levels of detail. Our protocol is structured to enable modular implementation of each of the described steps and enhance studies on embryogenesis, gene expression, or neurodevelopment.

Here, we present a combinatorial approach using high-resolution microscopy, computational tools, and single-cell labeling in living C. elegans embryos to understand single cell dynamics during neurodevelopment.

Isotrope Licht-Sheet-Mikroskopie und automatisierte Zelllineage-Analysen zu Katalog-Caenorhabditis elegans Embryogenese mit subzellulärer Auflösung

Caenorhabditis elegans (C. elegans) stands out as the only organism in which the challenge of understanding the cellular origins of an entire nervous ...

Entwicklungsbiologie

Measuring Sperm Guidance and Motility within the Caenorhabditis elegans Hermaphrodite Reproductive Tract

Successful fertilization is fundamental to sexual reproduction, yet little is known about the mechanisms that guide sperm to oocytes within the female reproductive tract. While in vitro studies suggest that sperm of internally fertilizing animals can respond to various cues from their surroundings, the inability to visualize their behavior inside the female reproductive tract creates a challenge for understanding sperm migration and mobility in its native environment. Here, we describe a method using C. elegans that overcomes this limitation and takes advantage of their transparent epidermis. C. elegans males stained with a mitochondrial&#160; dye are mated with adult hermaphrodites, which act as modified females, and deposit fluorescently labeled sperm into the hermaphrodite uterus. The migration and motility of the labeled sperm can then be directly tracked using an epi-fluorescence microscope in a live hermaphrodite. In wild-type animals, approximately 90% of the labeled sperm crawl through the uterus and reach the fertilization site, or spermatheca. Images of the uterus can be taken 1 h after mating to assess the distribution of the sperm within the uterus and the percentage of sperm that have reached the spermatheca. Alternatively, time-lapse images can be taken immediately after mating to assess sperm speed, directional velocity and reversal frequency. This method can be combined with other genetic and molecular tools available for the C.elegans to identify novel genetic and molecular mechanisms that are important in regulating sperm guidance and motility within the female reproductive tract.

Measuring Sperm Guidance and Motility within the Caenorhabditis elegans Hermaphrodite Reproductive Tract

Sperm must successfully navigate through the oviduct to fertilize an oocyte. Here, we describe an assay for measuring sperm migration within the C. elegans hermaphrodite uterus. This assay can provide quantitative data on sperm distribution within the uterus after mating, as well as on speed, directional velocity, and reversal frequency.

Messung der Spermienführung und-beweglichkeit innerhalb der Caenorhabditis elegans Hermaphrodite Reproductive Tract

Successful fertilization is fundamental to sexual reproduction, yet little is known about the mechanisms that guide sperm to oocytes within the female ...

A Semi-high-throughput Imaging Method and Data Visualization Toolkit to Analyze C. elegans Embryonic Development

C. elegans is the premier system for the systematic analysis of cell fate specification and morphogenetic events during embryonic development. One challenge is that embryogenesis dynamically unfolds over a period of about 13 h; this half day-long timescale has constrained the scope of experiments by limiting the number of embryos that can be imaged. Here, we describe a semi-high-throughput protocol that allows for the simultaneous 3D time-lapse imaging of development in 80&#8211;100 embryos at moderate time resolution, from up to 14 different conditions, in a single overnight run. The protocol is straightforward and can be implemented by any laboratory with access to a microscope with point visiting capacity. The utility of this protocol is demonstrated by using it to image two custom-built strains expressing fluorescent markers optimized to visualize key aspects of germ-layer specification and morphogenesis. To analyze the data, a custom program that crops individual embryos out of a broader field of view in all channels, z-steps, and timepoints and saves the sequences for each embryo into a separate tiff stack was built. The program, which includes a user-friendly graphical user interface (GUI), streamlines data processing by isolating, pre-processing, and uniformly orienting individual embryos in preparation for visualization or automated analysis. Also supplied is an ImageJ macro that compiles individual embryo data into a multi-panel file that displays maximum intensity fluorescence projection and brightfield images for each embryo at each time point. The protocols and tools described herein were validated by using them to characterize embryonic development following knock-down of 40 previously described developmental genes; this analysis visualized previously annotated developmental phenotypes and revealed new ones. In summary, this work details a semi-high-throughput imaging method coupled with a cropping program and ImageJ visualization tool that, when combined with strains expressing informative fluorescent markers, greatly accelerates experiments to analyze embryonic development.

A Semi-high-throughput Imaging Method and Data Visualization Toolkit to Analyze C. elegans Embryonic Development

This work describes a semi-high-throughput protocol that allows simultaneous 3D time-lapse imaging of embryogenesis in 80&#8211;100 C. elegans embryos in a single overnight run. Additionally, image processing and visualization tools are included to streamline data analysis. The combination of these methods with custom reporter strains enables detailed monitoring of embryogenesis.

Ein Instrument zur Analyse der C. elegans Embryonal development

C. elegans is the premier system for the systematic analysis of cell fate specification and morphogenetic events during embryonic development. One ...

4D Microscopy: Unraveling Caenorhabditis elegans Embryonic Development Using Nomarski Microscopy

4D microscopy is an invaluable tool for unraveling the embryonic developmental process in different animals. Over the last decades, Caenorhabditis elegans has emerged as one of the best models for studying development. From an optical point of view, its size and transparent body make this nematode an ideal specimen for DIC (Differential Interference Contrast or Nomarski) microscopy. This article illustrates a protocol for growing C. elegans nematodes, preparing and mounting their embryos, performing 4D microscopy and cell lineage tracing. The method is based on multifocal time-lapse records of Nomarski images and analysis with specific software. This technique reveals embryonic developmental dynamics at the cellular level. Any embryonic defect in mutants, such as problems in spindle orientation, cell migration, apoptosis or cell fate specification, can be efficiently detected and scored. Virtually every single cell of the embryo can be followed up to the moment the embryo begins to move. Tracing the complete cell lineage of a C. elegans embryo by 4D DIC microscopy is laborious, but the use of specific software greatly facilitates this task. In addition, this technique is easy to implement in the lab. 4D microscopy is a versatile tool and opens the possibility of performing an unparalleled analysis of embryonic development.

4D Microscopy: Unraveling Caenorhabditis elegans Embryonic Development Using Nomarski Microscopy

Here, we present a protocol for preparing and mounting Caenorhabditis elegans embryos, recording development under a 4D microscope and tracing cell lineage.

4D-Mikroskopie: Entschlüsselung der Caenorhabditis elegans Embryonalentwicklung mittels Nomarski-Mikroskopie

4D microscopy is an invaluable tool for unraveling the embryonic developmental process in different animals. Over the last decades, Caenorhabditis elegans ...

Imaging C. elegans Embryos using an Epifluorescent Microscope and Open Source Software

Cellular processes, such as chromosome assembly, segregation and cytokinesis,are inherently dynamic. Time-lapse imaging of living cells, using fluorescent-labeled reporter proteins or differential interference contrast (DIC) microscopy, allows for the examination of the temporal progression of these dynamic events which is otherwise inferred from analysis of fixed samples1,2. Moreover, the study of the developmental regulations of cellular processes necessitates conducting time-lapse experiments on an intact organism during development. The Caenorhabiditis elegans embryo is light-transparent and has a rapid, invariant developmental program with a known cell lineage3, thus providing an ideal experiment model for studying questions in cell biology4,5and development6-9. C. elegans is amendable to genetic manipulation by forward genetics (based on random mutagenesis10,11) and reverse genetics to target specific genes (based on RNAi-mediated interference and targeted mutagenesis12-15). In addition, transgenic animals can be readily created to express fluorescently tagged proteins or reporters16,17. These traits combine to make it easy to identify the genetic pathways regulating fundamental cellular and developmental processes in vivo18-21. In this protocol we present methods for live imaging of C. elegans embryos using DIC optics or GFP fluorescence on a compound epifluorescent microscope. We demonstrate the ease with which readily available microscopes, typically used for fixed sample imaging, can also be applied for time-lapse analysis using open-source software to automate the imaging process.

Imaging C. elegans Embryos using an Epifluorescent Microscope and Open Source Software

The C. elegans embryo is a powerful system for studying cell biology and development. We present a protocol for live imaging of C. elegans embryos utilizing DIC optics or fluorescence using readily available epifluorescent microscopes and open-source software.

Imaging C. elegans Embryos mit einem Epifluoreszenz-Mikroskop und Open Source Software

Cellular processes, such as chromosome assembly, segregation and cytokinesis,are inherently dynamic. Time-lapse imaging of living cells, using ...

Biologie

Basic Caenorhabditis elegans Methods: Synchronization and Observation

Research into the molecular and developmental biology of the nematode Caenorhabditis elegans was begun in the early seventies by Sydney Brenner and it has since been used extensively as a model organism 1. C. elegans possesses key attributes such as simplicity, transparency and short life cycle that have made it a suitable experimental system for fundamental biological studies for many years 2. Discoveries in this nematode have broad implications because many cellular and molecular processes that control animal development are evolutionary conserved 3.
C. elegans life cycle goes through an embryonic stage and four larval stages before animals reach adulthood. Development can take 2 to 4 days depending on the temperature. In each of the stages several characteristic traits can be observed. The knowledge of its complete cell lineage 4,5 together with the deep annotation of its genome turn this nematode into a great model in fields as diverse as the neurobiology 6, aging 7,8, stem cell biology 9 and germ line biology 10. 
An additional feature that makes C. elegans an attractive model to work with is the possibility of obtaining populations of worms synchronized at a specific stage through a relatively easy protocol. The ease of maintaining and propagating this nematode added to the possibility of synchronization provide a powerful tool to obtain large amounts of worms, which can be used for a wide variety of small or high-throughput experiments such as RNAi screens, microarrays, massive sequencing, immunoblot or in situ hybridization, among others. 
Because of its transparency, C. elegans structures can be distinguished under the microscope using Differential Interference Contrast microscopy, also known as Nomarski microscopy. The use of a fluorescent DNA binder, DAPI (4',6-diamidino-2-phenylindole), for instance, can lead to the specific identification and localization of individual cells, as well as subcellular structures/defects associated to them.

Basic Caenorhabditis elegans Methods: Synchronization and Observation

The easiness of maintaining and propagating the nematode C. elegans make it a nice model organism to work with. The possibility of synchronizing worms allows the work with a significant amount of subjects at the same developmental stage, what facilitates the study of one particular process in many animals.

Grundlegende<em&gt; Caenorhabditis elegans</em&gt; Methoden: Synchronisation und Observation

Research into the molecular and developmental biology of the nematode Caenorhabditis elegans was begun in the early seventies by Sydney Brenner and it has ...

Tracking and Quantifying Developmental Processes in C. elegans Using Open-source Tools

Quantitatively capturing developmental processes is crucial to derive mechanistic models and key to identify and describe mutant phenotypes. Here protocols are presented for preparing embryos and adult C. elegans animals for short- and long-term time-lapse microscopy and methods for tracking and quantification of developmental processes. The methods presented are all based on C. elegans strains available from the Caenorhabditis Genetics Center and on open-source software that can be easily implemented in any laboratory independently of the microscopy system used. A reconstruction of a 3D cell-shape model using the modelling software IMOD, manual tracking of fluorescently-labeled subcellular structures using the multi-purpose image analysis program Endrov, and an analysis of cortical contractile flow using PIVlab (Time-Resolved Digital Particle Image Velocimetry Tool for MATLAB) are shown. It is discussed how these methods can also be deployed to quantitatively capture other developmental processes in different models, e.g., cell tracking and lineage tracing, tracking of vesicle flow.

Tracking and Quantifying Developmental Processes in C. elegans Using Open-source Tools

Here it is shown how to track and quantify developmental processes in C. elegans. The methods presented are based on open-source tools that can be easily implemented. It is demonstrated how to reconstruct 3D cell-shape models, how to manually track subcellular structures, and how to analyze cortical contractile flow.

Tracking und Quantifizierung von Entwicklungsprozessen in<em&gt; C. elegans</em&gt; Verwendung von Open-Source-Werkzeuge

Quantitatively capturing developmental processes is crucial to derive mechanistic models and key to identify and describe mutant phenotypes. Here ...

Lifespan Analysis: Measuring C. elegans Longevity

Source: Cornwell, A. B., et al. <a href="https://www-jove-com.remotexs.ntu.edu.sg/video/57819/the-replica-set-method-high-throughput-approach-to-quantitatively">The Replica Set Method: A High-throughput Approach to Quantitatively Measure&#160;Caenorhabditis elegans&#160;Lifespan</a>.&#160;J. Vis. Exp.&#160; (2018).
This video describes a traditional, plate-based method of measuring the lifespan of C. elegans. The example protocol measures the lifespan of worms treated with RNAi.

Novel Metrics zur Charakterisierung von Embryonic Dehnung des Nematode

Zusammenfassung

Weitere Videos entdecken

Kapitel in diesem Video