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>
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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.

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-permeable. Consequently, when a large amount of bacteria (or embryos) is present on the pad, they may be deprived of oxygen after a few hours leading to their premature death. Having three-fold embryos &#8212; corresponding to embryos that are 3-fold in length compared to non-elongated embryos &#8212; recorded along with the embryos of interest will be a good indicator of potential hypoxic conditions, since those embryos will stop moving in their eggshells in the absence of oxygen. No morphological measurements should be done on hypoxic conditions or on dead embryos.
Another critical step when using time-lapse imaging is temperature at which development of the embryo occurs. Thermosensitive mutants are currently used in C. elegans. The biological effect of temperature shift may be immediate or delayed depending on the half-life of the protein and the nature of the mutation its carries. Consequently, the temperature at which the embryo is exposed should be constant over time and should be controlled by appropriate ventilation of the microscopy room or a heating-cooling chamber on the microscope stage.
Section 2 is dependent on larvae synchronization using alkaline hypochlorite treatment. Under certain circumstances, this treatment may lead to embryonic lethality (Emb). Emb above 20% in wt population suggests an elevated toxicity during hypochlorite treatment that may negatively impact morphogenesis. Synchronized L1 displaying high Emb in wt background should not be used for further analysis. This restriction also applies to protocol 3.
We do not recommend the use of anesthetic drugs to immobilize larvae. Levamisol in particular, immobilizes the nematode through induction of muscle tetany that tends to shrink the larvae introducing experimental bias. If exposure time is not quick enough as a result of the limitations of the microscope, we recommend reducing the motility of larvae by increasing the concentration of the agarose in the pad and reducing the amount of liquid between the pad and the coverslip. Care should be taken however, not to desiccate the larvae, since desiccation will reduce their size.
In section 3, measurement of the length of larvae used comparison of flow rates between analyzed samples. To do so, the distribution of control particles needs to be compared. If the distributions fully overlay, the TOF (time-of-flight) values obtained for corresponding samples can be compared, if not, these values need to be normalized. Normalization of sample-TOF over control particles-TOF (as detailed in 3.4.5.1) has been used successfully as shown for pak-1(ok448) (Figure 3C and Figure 5B). Relative length of pak-1(ok448) vs wt was found to be similar in at least 3 independent experiments with or without normalization (data not shown). However, we recommend, confirming the results obtained with normalization with those obtained without it, especially when comparing larvae with small size differences. It should be noted that measurement of the larvae length using flow-cytometry provides a length relative to a control sample, in this case, wt larvae rather than an absolute length in micrometer as for image analysis. This implies that measurements from independent experiments cannot be combined unless computing size ratio over wt.
The buffer used for dilution in the sample cup will have an impact on the flow rate. We observed that the sheath buffer recommended by the manufacturer contains detergent that increases the amount of bubbles generated during acquisition. Using M9 buffer, which does not contain detergent, significantly reduced the formation of bubbles but was less efficient in avoiding the plugging of eggs in the tubules of the sorter, which affects the flow rate of samples. Egg plugging is easily detected during the acquisition by a marked decrease of the observable TOF of control particles for a few seconds followed by an elevated TOF- also for a few seconds. Egg plugging may also lead to the obstruction of the channel and the complete arrest of the particle flow (less that 5 objects per second). If this should occur, click on the CLEAN button until flow rate is restored. Any measurements occurring during these events should be excluded from the data analysis. Sheath buffer may be recommended for experiments involving strains characterized by high rates of dead eggs.
The sample and sheath pressure (set at the step 3.2.3), may change (slightly) over time and should be adjusted manually throughout the experiment in order to ensure a very constant flow rate. Reduction or increase of the low rate will be observable when analyzing the results and plotting the TOFs of control particles over time (Figure 3C). Reduction of the flow rate will result in the increase of the average TOFs of particles over time, while an increase of the flow rate will result in the opposite. Alteration of the flow rate will negatively impact the sensitivity of the method in detecting small size differences between populations of larvae.
Methods aiming to identify genes controlling elongation and requiring time-lapse microscopy recording are generally highly time-consuming and tedious when phenotyping several genotypes. The flow-cytometer-based approach, while requiring equipment not available to all laboratories, is less time-consuming and consequently more efficient when several strains need to be characterized. This method is also more statistically robust compared to measurements using image analysis (assessed by the student&#39;s t-test comparing mutant and wt TOF distributions; Figure 5A and B). This method may then be highly suitable for strains expressing elongation defects with low expressivity/penetrance.
Several methods using flow-cytometry have been developed in the past to measure fitness of nematodes9-12. These methods use animals dispensed in a 96-well plates and the Reflex module of the worm sorter&#39;. The Reflex module enables direct analysis of nematode population dispensed within 96-well plates. Consequently, these methods are able to characterize hundreds of conditions per day and constitute a robust manner in which to measure the fitness of a non-synchronized population. They are however, not well suited to identify small size differences between synchronized L1. Measurement of small differences between L1 larvae requires the measurement across a large number of animals, which is incompatible with the use of 96-well plates and of the Reflex module that may efficiently characterize 100 objects at the most per well. The method described here is designed for this purpose at the expense of the throughput, which is significantly reduced. It enables the characterization of 3 to 4 conditions per hour once the instrument is calibrated, which is a marked improvement over using image analysis in protocol 2.
Measurement of head and tail width ratio is the first method that was developed to characterize morphogenic processes occurring unevenly along the antero-posterior axis of the embryo7. When applied to genes shown to control early elongation, these methods will clarify the spatial distribution of signaling pathways controlling morphogenesis at that stage. Measurement of the length of arrested larvae using either image analysis or flow cytometry in combination with measurement of the length of the embryos at the end of early elongation will enable the identification of genes controlling either early or late elongation or both with high sensitivity and precision. These procedures may then contribute significantly to future understanding of the spatial and temporal regulation of signaling pathways controlling embryonic elongation in C. elegans. Furthermore, these approaches can also be adapted to study signaling pathways controlling body length such as the insulin and TGF-beta pathways 13,14 and chronic exposure to environmental contaminants 15,16. These measurements can be done at different larval stages or in adults using minor variations of Protocols 2 and 3. Measuring size differences in L1 is more challenging with the worm sorter than larger objects such as L3, L4 larvae or adults. Protocol 3 can then easily be adapted to do these measurements.

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 pak-1 code respectively for a guanine-exchange factor (GEF) and an effector specific for Rac/Cdc42 GTPases and let-502 codes for an effector for RHO-1 7. In this study,pix-1 and pak-1 were shown to control epidermal morphogenesis during early elongation7. This study used head and tail width measurement as metrics demonstrate for the first time, that hypodermal cells located in the anterior embryo follow different morphogenic programs than those located in its posterior 7. To establish the reproducibility and robustness of these metrics, head width was measured independently five times on 12 wild-type (wt) embryos at 1.2-fold stage. Variances among the five different groups of measurement were compared then assessed using the Brown-Forsythe test (using R statistical package) revealing no significant differences amongst the measurements (F-test p-value &#62; 0.5; Figure 4A) suggesting that measurements for a group of embryos are reproducible. Assessment of reproducibility and batch effects associated with these measurements was done through measurement of the head width of wt embryos at 1.2-fold stage from 4D-DIC imaging done on three different days (n = 12 embryos). Across these three measurement groups, the variance was not significantly different and no significant batch effects were found to impact the head-width measurement results (F-test p-value &#62; 0.5; Figure 4B).
Similar results were obtained for tail width measurements and for measurements done at different stages of early elongation (data not shown). These data established head-width, tail-width and head/tail width ratio as robust metrics to characterize early elongation defects.
Measuring Head and Tail Width as well as Length of the Embryos Allows for the Characterization of Genes Controlling Early Elongation along the Antero-posterior Axis of the Embryo.
Measurement of the length of the embryos, as well as the width of their head and tail was done on wt and mutant embryos carrying null or thermosensitive strong loss-of function alleles for genes controlling early elongation: pix-1(gk416); pak-1(ok448) and let-502(sb118ts)7. We measured the head/tail (H/T) width ratio of wt and mutant embryos at the beginning (1.2-fold stage) and at the end of the early elongation at non-permissive temperatures (Figure 4C left and right panel respectively). While at the beginning of early elongation there was no change- or a reduced H/T ratio was observed in pix-1, pak-1 and let-502 mutants when compared to wt animals (1.2-fold stage, Figure 2C, left panel), at the end of early elongation all three mutants showed a significantly higher H/T ratio than wt embryos (t-test p-values &#60; 0.006; Figure 4C; right panel). This demonstrated that pix-1, pak-1 and let-502 mutant embryos display abnormal antero-posterior morphology at the end of early elongation. Further analysis comparing head width and tail width between 1.2-fold stage and the end of early elongation, using the same measurement parameters revealed that the head width is less reduced in pix-1, pak-1 and let-502 mutants while the tail width reduces significantly less in let-502 mutants only 7. This revealed that let-502 controls morphogenic processes similarly along the antero-posterior axis of the embryo, while pix-1 and pak-1 control morphogenic processes occurring mainly at the anterior part of the embryo7. The length difference between the embryos at the end versus the beginning of early elongation (Figure 4D) was also measured. We found that early elongation was significantly reduced in pix-1, pak-1 and let-502 mutants when compared to wt suggesting that alteration of the anterior morphogenesis in pix-1 and pak-1 mutants alone is sufficient to significantly reduce the elongation of the embryo.
Protocol 2 and 3 were used to assess the length of arrested larvae in wt and mutant backgrounds. The length of these larvae was assessed using both image analysis (Protocol 2)7 and flow-cytometry (Protocol 3; unpublished data). Measurements of larvae length using image analysis results in absolute measurements of animals&#39; length in micrometers in a robust and highly reproducible manner (Figure 5A). This analysis revealed that synchronized mutant larvae display significantly reduced length when compared to wt (Figure 5A)7. Measurement of these larvae using the flow-cytometry based protocol (Protocol 3) gave comparable results (Figure 5B). However, it should be noted that the large number of larvae measured using the latter approach significantly increased the statistical robustness of genotype comparison (t-test p-values &#60;10-24). Based on these findings, the flow-cytometry approach may be a better choice over image analysis in order to characterize mutant animals displaying very subtle elongation defects.
<img alt="Figure 1" src="/files/ftp_upload/53712/53712fig1.jpg" /> 
Figure 1. Preparation of an Agarose Pad. A, Microscope slide placed between two spacer-slides covered by two layers of masking tape. B, The agarose pad is covered with another slide supported by the two spacer-slides. C, The final shape and size of the agarose pad after cutting with a razor blade to fit the coverslip. <a href="https://www.jove.com/files/ftp_upload/53712/53712fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/53712/53712fig2.jpg" /> 
Figure 2. Measurement of Early and Late Elongation Defects. A - B, Measurement of the length of an embryo at comma stage (A) and at the end of early elongation (B). The red line, used to measure the embryos was drawn using the segmented line tool of ImageJ. C, Head and tail width are measured in embryos at 1.2-fold stage (left) and at the end of early elongation (right). Arrows represent the localization of measured areas (modified from Martin et al., 2014). Scale bar: 20 &#956;m. D, Length of Larvae is measured for synchronized L1-larvae. Right panel is an enlarged view of the captured image (left). The red line was drawn using the segmented line tool of ImageJ. It is used to measure the length of the larva. Scale bar: 100 &#956;m. <a href="https://www.jove.com/files/ftp_upload/53712/53712fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" src="/files/ftp_upload/53712/53712fig3.jpg" /> 
Figure 3. Measurement of the Length of Synchronized Larvae Using Flow-cytometry. A, Gating and sorting window of COPAS Biosort showing Green and Red emission of control particles, bubbles, dead and living animals with respect to their Time-Of-Flight (TOF). B, TOF of control particles at different time points for a representative experiment. The slope of the linear function of TOF versus time is around 10-5, indicating that TOF of control particles is constant over time throughout the experiment. C, Distribution of control particle TOFs expressed as a percentage of maximal value of distributions. Non-normalized and normalized control particle distributions are represented for pak-1(ok448). Other distributions are not normalized. D, distribution of TOFs for living animals expressed as a percentage of the maximal value of the distributions. TOFs are not normalized on control particles except for pak-1(ok448) as indicated. <a href="https://www.jove.com/files/ftp_upload/53712/53712fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" src="/files/ftp_upload/53712/53712fig4.jpg" /> 
Figure 4. Pix-1, Pak-1 and Let-502 Mutants Present Early Elongation Defects. A - B, Reproducibility and robustness assessment of head width measurements. A, Five independent measurements of the head width for wt embryos at 1.2-fold stage (n = 12 embryos). Means and standard deviations (error bars) are indicated. Non significant (ns) differences between measurements were computed using the Brown-Forsythe test (using R statistical package) (F-test p-value &#62; 0.5). B, Head width measurement for wt embryos at three different days (n = 12 embryos). Means and standard deviations are indicated as well; there was no significant difference in variance across the measurements (ns; Brown-Forsythe F-test p-value &#62; 0.5). C, Distributions of head/tail width ratio at 1.2-fold stage (left panel), at the end of early elongation (right panel) in wt, pix-1(gk416), pak-1(ok448) and let-502(sb118ts) mutants at 23 - 24 &#176;C. Note that mothers of let-502ts embryos used for this study were grown at 25.5 &#176;C. D, Distribution of the elongation in wt, pix-1(gk416), pak-1(ok448) and let-502(sb118ts) mutants between comma stage and the start of late elongation. The box-plots represent the min, max, 25th, 50th (median) and 75th percentiles of the populations. * t-test p-value &#60; 0.05, ** t-test p-value &#60; 0.006 (modified from Martin et al., 2014). <a href="https://www.jove.com/files/ftp_upload/53712/53712fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" src="/files/ftp_upload/53712/53712fig5.jpg" /> 
Figure 5. Pix-1(gk416), Pak-1(ok448) and Let-502(sb118ts) Larvae&#160;Present Length Defects. A, Length of larvae measured in wt, pix-1(gk416), pak-1(ok448) and let-502(sb118ts) animals measured using image analysis (Protocol 2). B, Relative larvae length measured using flow-cytometer (Protocol 3). Numbers in brackets correspond to the number of animals used for the measurements. Means of lengths and standard error of the mean (SEM; error bars) are represented. Student&#39;s t-test p-values are indicated (p). <a href="https://www.jove.com/files/ftp_upload/53712/53712fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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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Universidad San Sebastian

Research

JoVE Journal

Neuroscience

6.0K Views.  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. 

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

Multi-unit Recording Methods to Characterize Neural Activity in the Locust (Schistocerca Americana) Olfactory Circuits

Detection and interpretation of olfactory cues are critical for the survival of many organisms. Remarkably, species across phyla have strikingly similar olfactory systems suggesting that the biological approach to chemical sensing has been optimized over evolutionary time1. In the insect olfactory system, odorants are transduced by olfactory receptor neurons (ORN) in the antenna, which convert chemical stimuli into trains of action potentials. Sensory input from the ORNs is then relayed to the antennal lobe (AL; a structure analogous to the vertebrate olfactory bulb). In the AL, neural representations for odors take the form of spatiotemporal firing patterns distributed across ensembles of principal neurons (PNs; also referred to as projection neurons)2,3. The AL output is subsequently processed by Kenyon cells (KCs) in the downstream mushroom body (MB), a structure associated with olfactory memory and learning4,5. Here, we present electrophysiological recording techniques to monitor odor-evoked neural responses in these olfactory circuits.
First, we present a single sensillum recording method to study odor-evoked responses at the level of populations of ORNs6,7. We discuss the use of saline filled sharpened glass pipettes as electrodes to extracellularly monitor ORN responses. Next, we present a method to extracellularly monitor PN responses using a commercial 16-channel electrode3. A similar approach using a custom-made 8-channel twisted wire tetrode is demonstrated for Kenyon cell recordings8. We provide details of our experimental setup and present representative recording traces for each of these techniques.

Multi-unit Recording Methods to Characterize Neural Activity in the Locust Schistocerca Americana Olfactory Circuits

We demonstrate variations of the extracellular multi-unit recording technique to characterize odor-evoked responses in the first three stages of the invertebrate olfactory pathway. These techniques can easily be adapted to examine ensemble activity in other neural systems as well.

Detection and interpretation of olfactory cues are critical for the survival  of many organisms. Remarkably, species across phyla have strikingly similar ...

A Caenorhabditis elegans Model System for Amylopathy Study

Amylopathy is a term that describes abnormal synthesis and accumulation of amyloid beta (A&beta;) in tissues with time. A&beta; is a hallmark of Alzheimer's disease (AD) and is found in Lewy body dementia, inclusion body myositis and cerebral amyloid angiopathy 1-4. Amylopathies progressively develop with time. For this reason simple organisms with short lifespans may help to elucidate molecular aspects of these conditions. Here, we describe experimental protocols to study A&beta;-mediated neurodegeneration using the worm Caenorhabditis elegans. Thus, we construct transgenic worms by injecting DNA encoding human A&beta;42 into the syncytial gonads of adult hermaphrodites. Transformant lines are stabilized by a mutagenesis-induced integration. Nematodes are age synchronized by collecting and seeding their eggs. The function of neurons expressing A&beta;42 is tested in opportune behavioral assays (chemotaxis assays). Primary neuronal cultures obtained from embryos are used to complement behavioral data and to test the neuroprotective effects of anti-apoptotic compounds.

A Caenorhabditis elegans Model System for Amylopathy Study

We describe methods to study aspects of amylopathies in the worm C. elegans. We show how to construct worms expressing human A&beta;42 in neurons and how to test their function in behavioral assays. We further show how to obtain primary neuronal cultures that can be used for pharmacological testing.

Amylopathy is a term that describes abnormal synthesis and accumulation of amyloid beta (A&beta;) in tissues with time. A&beta; is a hallmark of ...

Methods for Studying the Mechanisms of Action of Antipsychotic Drugs in Caenorhabditis elegans

Caenorhabditis elegans is a simple genetic organism amenable to large-scale forward and reverse genetic screens and chemical genetic screens. The C. elegans genome includes potential antipsychotic drug (APD) targets conserved in humans, including genes encoding proteins required for neurotransmitter synthesis and for synaptic structure and function. APD exposure produces developmental delay and/or lethality in nematodes in a concentration-dependent manner. These phenotypes are caused, in part, by APD-induced inhibition of pharyngeal pumping1,2. Thus, the developmental phenotype has a neuromuscular basis, making it useful for pharmacogenetic studies of neuroleptics. Here we demonstrate detailed procedures for testing APD effects on nematode development and pharyngeal pumping. For the developmental assay, synchronized embryos are placed on nematode growth medium (NGM) plates containing APDs, and the stages of developing animals are then scored daily. For the pharyngeal pumping rate assay, staged young adult animals are tested on NGM plates containing APDs. The number of pharyngeal pumps per unit time is recorded, and the pumping rate is calculated. These assays can be used for studying many other types of small molecules or even large molecules.

Methods for Studying the Mechanisms of Action of Antipsychotic Drugs in Caenorhabditis elegans

Approaches for testing the effects of antipsychotic drugs (APDs) in Caenorhabditis elegans are demonstrated. Assays are described for testing drug effects on development and viability and on pharyngeal pumping rate. These methods are also applicable for pharmacogenetic experiments with drug classes other than APDs.

Caenorhabditis elegans is a simple genetic organism amenable to  large-scale forward and reverse genetic screens and chemical genetic screens. The C.  ...

An Assay for Measuring the Effects of Ethanol on the Locomotion Speed of Caenorhabditis elegans

Alcohol use disorders are a significant public health concern, for which there are few effective treatment strategies. One difficulty that has delayed the development of more effective treatments is the relative lack of understanding of the molecular underpinnings of the effects of ethanol on behavior. The nematode, Caenorhabditis elegans (C.&#160;elegans), provides a useful model in which to generate and test hypotheses about the molecular effects of ethanol. Here, we describe an assay that has been developed and used to examine the roles of particular genes and environmental factors in behavioral responses to ethanol, in which locomotion is the behavioral output. Ethanol dose-dependently causes an acute depression of crawling on an agar surface. The effects are dynamic; animals exposed to a high concentration demonstrate an initial strong depression of crawling, referred to here as initial sensitivity, and then partially recover locomotion speed despite the continued presence of the drug. This ethanol-induced behavioral plasticity is referred to here as the development of acute functional tolerance. This assay has been used to demonstrate that these two phenotypes are distinct and genetically separable. The straightforward locomotion assay described here is suitable for examining the effects of both genetic and environmental manipulations on these acute behavioral responses to ethanol in C.&#160;elegans.

An Assay for Measuring the Effects of Ethanol on the Locomotion Speed of Caenorhabditis elegans

C. elegans is a useful model for studying the effects of ethanol on behavior. We present a behavioral assay that quantifies the effects of ethanol on the locomotion speed of crawling worms; both initial sensitivity and the development of acute functional tolerance to ethanol can be measured with this assay.

Alcohol use disorders are a significant public health concern, for which there are few effective treatment strategies. One difficulty that has delayed the ...

Agarose Microchambers for Long-term Calcium Imaging of Caenorhabditis elegans

Behavior is controlled by the nervous system. Calcium imaging is a straightforward method in the transparent nematode Caenorhabditis elegans to measure the activity of neurons during various behaviors. To correlate neural activity with behavior, the animal should not be immobilized but should be able to move. Many behavioral changes occur during long time scales and require recording over many hours of behavior. This also makes it necessary to culture the worms in the presence of food. How can worms be cultured and their neural activity imaged over long time scales? Agarose Microchamber Imaging (AMI) was previously developed to culture and observe small larvae and has now been adapted to study all life stages from early L1 until the adult stage of C. elegans. AMI can be performed on various life stages of C. elegans. Long-term calcium imaging is achieved without immobilizing the animals by using short externally triggered exposures combined with an electron multiplying charge-coupled device (EMCCD) camera recording. Zooming out or scanning can scale up this method to image up to 40 worms in parallel. Thus, a method is described to image behavior and neural activity over long time scales in all life stages of C. elegans.

Agarose Microchambers for Long-term Calcium Imaging of Caenorhabditis elegans

Imaging behavior and neural activity over long time scales without immobilization of the animal is a prerequisite to understand behavior. Agarose microfluidic chambers imaging (AMI) can be used to image neural activity and behavior for all life stages of Caenorhabditis elegans.

Behavior is controlled by the nervous system. Calcium imaging is a straightforward method in the transparent nematode Caenorhabditis elegans to measure ...

Dynamic Quantitative Sensory Testing to Characterize Central Pain Processing

Central facilitation and modulation of incoming nociceptive signals play an important role in the perception of pain. Disruption in central pain processing is present in many chronic pain conditions and can influence responses to specific therapies. Thus, the ability to precisely describe the state of central pain processing has profound clinical significance in both prognosis and prediction. Because it is not practical to record neuronal firings directly in the human spinal cord, surrogate behavior tests become an important tool to assess the state of central pain processing. Dynamic QST is one such test, and can probe both the ascending facilitation and descending modulation of incoming nociceptive signals via TS and CPM, respectively. Due to the large between-individual variability in the sensitivity to noxious signals, standardized TS and CPM tests may not yield any meaningful data in up to 50% of the population due to floor or ceiling effects. We present methodologies to individualize TS and CPM so we can capture these measures in a broader range of individuals than previously possible. We have used these methods successfully in several studies at the lab, and data from one ongoing study will be presented to demonstrate feasibility and potential applications of the methods.

By assessing pain in response to repetitive or different types of standardized stimuli, dynamic quantitative sensory testing (QST) can reveal changes in the central processing of pain. We present methods to optimize and individualize two dynamic QST measures: temporal summation (TS) and conditioned pain modulation (CPM).

Central facilitation and modulation of incoming nociceptive signals play an important role in the perception of pain. Disruption in central pain ...

A Caenorhabditis elegans Nutritional-status Based Copper Aversion Assay

To ensure survival, organisms must be capable of avoiding unfavorable habitats while ensuring a consistent food source. Caenorhabditis elegans alter their locomotory patterns upon detection of diverse environmental stimuli and can modulate their suite of behavioral responses in response to starvation conditions. Nematodes typically exhibit a decreased aversive response when removed from a food source for over 30 min. Observation of behavioral changes in response to a changing nutritional status can provide insight into the mechanisms that regulate the transition from a well-fed to starved state.
We have developed an assay that measures a nematode&#39;s ability to cross an aversive barrier (i.e. copper) then reach a food source over a prolonged period of time. This protocol builds upon previous work by integrating multiple variables in a manner that allows for continued data collection as the organisms shift towards an increasingly starved condition. Moreover, this assay permits an increased sample size so that larger populations of nematodes can be simultaneously evaluated.
Organisms defective for the ability to detect or respond to copper immediately cross the chemical barrier, while wild type nematodes are initially repelled. As wild type worms are increasingly starved, they begin to cross the barrier and reach the food source. We designed this assay to evaluate a mutant that is incapable of responding to diverse environmental cues, including food sensation or detection of aversive chemicals. When evaluated via this protocol, the defective organisms immediately crossed the barrier, but were also incapable of detecting a food source. Hence, these mutants repeatedly cross the chemical barrier despite temporarily reaching a food source. This assay can straightforwardly test populations of worms to evaluate potential pathway defects related to aversion and starvation.

A Caenorhabditis elegans Nutritional-status Based Copper Aversion Assay

Here, we present a Caenorhabditis elegans-specific assay designed to evaluate changes in copper aversion behavior and the ability to locate a common food source, as the organism progresses from a well-fed to starved nutritional state.

To ensure survival, organisms must be capable of avoiding unfavorable habitats while ensuring a consistent food source. Caenorhabditis elegans alter their ...

Ratiometric Calcium Imaging of Individual Neurons in Behaving Caenorhabditis Elegans

It has become increasingly clear that neural circuit activity in behaving animals differs substantially from that seen in anesthetized or immobilized animals. Highly sensitive, genetically encoded fluorescent reporters of Ca2+ have revolutionized the recording of cell and synaptic activity using non-invasive optical approaches in behaving animals. When combined with genetic and optogenetic techniques, the molecular mechanisms that modulate cell and circuit activity during different behavior states can be identified.
Here we describe methods for ratiometric Ca2+ imaging of single neurons in freely behaving Caenorhabditis elegans worms. We demonstrate a simple mounting technique that gently overlays worms growing on a standard Nematode Growth Media (NGM) agar block with a glass coverslip, permitting animals to be recorded at high-resolution during unrestricted movement and behavior. With this technique, we use the sensitive Ca2+ reporter GCaMP5 to record changes in intracellular Ca2+ in the serotonergic Hermaphrodite Specific Neurons (HSNs) as they drive egg-laying behavior. By co-expressing mCherry, a Ca2+-insensitive fluorescent protein, we can track the position of the HSN within ~ 1 &#181;m and correct for fluctuations in fluorescence caused by changes in focus or movement. Simultaneous, infrared brightfield imaging allows for behavior recording and animal tracking using a motorized stage. By integrating these microscopic techniques and data streams, we can record Ca2+ activity in the C. elegans egg-laying circuit as it progresses between inactive and active behavior states over tens of minutes.

Ratiometric Calcium Imaging of Individual Neurons in Behaving Caenorhabditis Elegans

This protocol describes the use of genetically encoded Ca2+ reporters to record changes in neural activity in behaving Caenorhabditis elegans worms. &#8195;

It has become increasingly clear that neural circuit activity in behaving animals differs substantially from that seen in anesthetized or immobilized ...

Combining Optogenetics with Artificial microRNAs to Characterize the Effects of Gene Knockdown on Presynaptic Function within Intact Neuronal Circuits

The purpose of this protocol is to characterize the effect of gene knockdown on presynaptic function within intact neuronal circuits. We describe a workflow on how to combine artificial microRNA (miR)-mediated RNA interference with optogenetics to achieve selective stimulation of manipulated presynaptic boutons in acute brain slices. The experimental approach involves the use of a single viral construct and a single neuron-specific promoter to drive the expression of both an optogenetic probe and artificial miR(s) against presynaptic gene(s). When stereotactically injected in the brain region of interest, the expressed construct makes it possible to stimulate with light exclusively the neurons with reduced expression of the gene(s) under investigation. This strategy does not require the development and maintenance of genetically modified mouse lines and can in principle be applied to other organisms and to any neuronal gene of choice. We have recently applied it to investigate how the knockdown of alternative splice isoforms of presynaptic P/Q-type voltage-gated calcium channels (VGCCs) regulates short-term synaptic plasticity at CA3 to CA1 excitatory synapses in acute hippocampal slices. A similar approach could also be used to manipulate and probe the neuronal circuitry in vivo.

This protocol provides a workflow on how to combine artificial microRNA-mediated RNA interference with optogenetics to stimulate specifically presynaptic boutons with reduced expression of selective gene(s) within intact neuronal circuits.

The purpose of this protocol is to characterize the effect of gene knockdown on presynaptic function within intact neuronal circuits. We describe a ...

Imaging Dendritic Spines in Caenorhabditis elegans

Dendritic spines are specialized sites of synaptic innervation modulated by activity and serve as substrates for learning and memory. Recently, dendritic spines have been described for DD GABAergic neurons as the input sites from presynaptic cholinergic neurons in the motor circuit of Caenorhabditis elegans. This synaptic circuit can now serve as a powerful new in vivo model of spine morphogenesis and function that exploits the facile genetics and ready accessibility of C. elegans to live-cell imaging. 
This protocol describes experimental strategies for assessing DD spine structure and function. In this approach, a super-resolution imaging strategy is used to visualize the intricate shapes of actin-rich dendritic spines. To evaluate the DD spine function, the light-activated opsin, Chrimson, stimulates the presynaptic cholinergic neurons, and the calcium indicator, GCaMP, reports the evoked calcium transients in postsynaptic DD spines. Together, these methods comprise powerful approaches for identifying genetic determinants of dendritic spines in C. elegans that could also direct spine morphogenesis and function in the brain.

Imaging Dendritic Spines in Caenorhabditis elegans

Dendritic spines are important cellular features of the nervous system. Here live imaging methods are described for assessing the structure and function of dendritic spines in C. elegans.&#160;These approaches support the development of mutant screens for genes that define dendritic spine shape or function.

Dendritic spines are specialized sites of synaptic innervation modulated by activity and serve as substrates for learning and memory. Recently, dendritic ...