Name: novel metrics to characterize embryonic elongation of the nematode caenorhabditis elegans
Uploaded: 2016-03-28T13:00:00
Description: Watch this Scientific Journal Video about Novel Metrics to Characterize Embryonic Elongation of the Nematode Caenorhabditis elegans at JoVE.com

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).

1. Characterization of Early Elongation Defects in WT and Mutant Animals
<ol>
	<li>Mounting Embryos for Normarski DIC Microscopy
	<ol>
		<li>Prepare the following culture media and material:
		<ol>
			<li>M9 Buffer, dissolve 12.8 g/L Na2HPO4&#8226;7H2O, 3 g/L KH2PO4, 5 g/L NaCl, 0.25 g/L MgSO4&#8226;7H2O in distilled water and autoclave to sterilize.</li>
			<li>NGM plates, dissolve 3 g/L NaCl, 16 g/L agar, 2.5 g/L ba...

<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>

This protocol describes novel metrics to characterize early and late phases of embryonic elongation.
In section 1, the critical step is the potential presence of bacteria on the pad. The embryos are hermetically enclosed between the pad and the coverslip during image acquisition. Sealing the slide is required to avoid desiccation of the animals during acquisition that lasts more than two hours. To our knowledge, none of the sealers used to mount agarose pads between slide and coverslip are air...

For nearly 50 years the nematode Caenorhabditis elegans established itself as a powerful model to study important questions in development, neurobiology, evolution, host-pathogen interactions, etc.1 The strength of this model in the study of development lies in: its short life cycle of 3 days; the ease with which these animals can be genetically altered; its transparency that enables the observation of cell displacement and morphology in living animals and its development that is mostly extra-uterine. The developmental stages of the nematode involve embryogenesis and four larval stages (L1 to L4), followed by adulthood. During embryonic development, epidermal morphogenesis drew considerable attention for its ability to enable a better understanding of how epithelial cells migrate as a group, how they reorganize their junctions and modify their individual morphology as well as their relative positioning within a functional epithelium. Epidermal morphogenesis is divided into four stages: dorsal intercalation consisting in the reorganization of dorsal epidermal cells, referred to as the hypodermis; ventral enclosure, consisting in migration of ventral hypodermal cells towards the ventral midline thus encasing the embryo in an epithelial cell monolayer; early and late elongation transforming the bean-shaped embryo into vermiform larvae. Following morphogenesis, embryo hatch and L1 larvae start feeding using available bacteria in their immediate environment.
Embryonic elongation is therefore a late phase of the embryonic development. It consists of the extension of the embryo along its longitudinal axis and a reduction of its transverse diameter. This involves a dramatic modification of the shape of the hypodermal cells. Elongation is divided into an early and a late phase. The early phase starts at the comma stage and ends when body-wall muscles start contracting at 1.75-fold stage in wild-type (wt) embryos &#8212; corresponding to embryos that are 1.75-fold in length compared to non-elongated embryos. Morphogenic processes occurring at that stage are mainly driven by contraction of filamentous actin bundles (FBs) located at the apical pole of hypodermal cells that drive their elongation along the antero-posterior axis of the embryo2. Contraction of FBs is control by phosphorylation of myosin-light chains by three kinases LET-502/ROCK, MRCK-1 and PAK-1 5. The late phase of the elongation, starts when body-wall muscles become functional and start contracting. It involves mechanotransduction signaling from the body-wall muscles to the dorsal and ventral hypodermal cells and ends when animals hatch3.
Elongation defects are generally characterized by the percentage of animals dying as embryos (Embryonic lethality; Emb) and those arresting their development as L1 larvae (Larval arrest phenotype; Lva) and being significantly shorter than wt. Identification of the stage of developmental arrest requires microscopic observation of dead embryos and arrested Larvae3-6.
It was recently shown that several genes, such as the Cdc42/Rac regulator and effector pix-1 and pak-1, control morphogenic processes during both early and late elongation3,7. We also recently showed that morphogenic processes differ along the antero-posterior axis of the embryos during early elongation37. These findings motivated the development of novel metrics specifically targeting early or late elongation stages and other metrics enabling the characterization of the morphology of embryos along their antero-posterior axis during early elongation.
These novel methods consist in measuring the length of embryos at the beginning and at the end of early elongation as well as the width of their heads and tails.7 Two protocols were also developed to measure the length of newly hatched larvae, synchronized at L1 stage7.
The eggshells of the embryos protect them against alkaline hypochlorite treatment while larvae, adults and bacteria present in the culture media are dissolved by the treatment. This treatment is then used to purify embryos from a non-synchronized population containing a majority of well-fed adults8. Food restriction is used to synchronize newly hatched larvae. Measuring the length of these larvae is then used to detect elongation defects. This measurement is preferred over the measurement of arrested larvae on culture plates because larvae that hatch from non-fully elongated embryos can recover to &#34;normal length&#34; when feeding but will maintain their reduced size when arrested in the absence of food.
Here, we present detailed protocols enabling the measurement of the length of elongating embryos as well as the width of their head and tail using time-lapse DIC microscopy and image analysis (Protocol 1). We also provide detailed protocols to measure the length of synchronized larvae using image analysis (Protocol 2) and Flow-Cytometry (Protocol 3).

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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-, Tail- and Head/Tail-width Ratio are Robust Metrics.
The protocols described here have been successfully used to characterize the function of regulators and effectors of Rho GTPases pix-1, pak-1 and let-502 during early elongation7. pix-1 and ...

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

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.

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. ...

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