Name: a semi-high-throughput imaging method and data visualization toolkit to analyze c. elegans embryonic development
Uploaded: 2019-10-29T15:00:00
Description: Watch this Scientific Journal Video about A Semi-high-throughput Imaging Method and Data Visualization Toolkit to Analyze C. elegans Embryonic Development at JoVE.com

S.D.O. was supported by the National Institute of General Medical Sciences-sponsored University of California San Diego Institutional Research and Academic Career Development Award (NIH/IRACDA K12 GM068524). A.D. and K.O. were supported by the Ludwig Institute for Cancer Research, which also provided them with research funding used to support this work. We are grateful to Andrew Chisholm for his advice in the early phases of this project, Ronald Biggs for contributions to this project after the initial method development phase, and Dave Jenkins and Andy Shiau for support and access to the Small Molecule Discovery group&#8217;s high-content imaging system.

1. Preparing C. elegans Embryos for Semi-high-throughput Imaging
NOTE: The goal of this portion of the protocol is to load a population of semi-synchronized (2 to 8-cell stage) C. elegans embryos, dissected from suitable marker strains (Figure 2), into a glass-bottom 384-well plate for imaging. Other plate formats could also work, but the 384 well plates are preferred because the small well size constrains the spread of embryos to ...

<ol>
	<li>Armenti, S. T., Nance, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adherens+junctions+in+C.+elegans+embryonic+morphogenesis.">Adherens junctions in C. elegans embryonic morphogenesis.</a> Sub-Cellular Biochemistry. 60, 279-299 (2012).</li><li>Chisholm, A. D., Hsiao, T. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Caenorhabditis+elegans+epidermis+as+a+model+skin.+I:+development,+patterning,+and+growth.">The Caenorhabditis elegans epidermis as a model skin. I: development, patterning, and growth.</a> Wiley Interdisciplinary Reviews Developmental Biology. 1 (6), 861-878 (2012).</li><li>Jackson, B. M., Eisenmann, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=beta-catenin-dependent+Wnt+signaling+in+C.+elegans:+teaching+an+old+dog+a+new+trick.">beta-catenin-dependent Wnt signaling in C. elegans: teaching an old dog a new trick.</a> Cold Spring Harbor Perspectives In Biology. 4 (8), 007948 (2012).</li><li>Lamkin, E. R., Heiman, M. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Coordinated+morphogenesis+of+neurons+and+glia.">Coordinated morphogenesis of neurons and glia.</a> Current Opinion in Neurobiology. 47, 58-64 (2017).</li><li>Loveless, T., Hardin, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cadherin+complexity:+recent+insights+into+cadherin+superfamily+function+in+C.+elegans.">Cadherin complexity: recent insights into cadherin superfamily function in C. elegans.</a> Current Opinion in Cell Biology. 24 (5), 695-701 (2012).</li><li>Priess, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Notch+signaling+in+the+C.+elegans+embryo.">Notch signaling in the C. elegans embryo.</a> WormBook. , 1-16 (2005).</li><li>Spickard, E. A., Joshi, P. M., Rothman, J. 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+multipotency-to-commitment+transition+in+Caenorhabditis+elegans-implications+for+reprogramming+from+cells+to+organs.">The multipotency-to-commitment transition in Caenorhabditis elegans-implications for reprogramming from cells to organs.</a> FEBS Letters. 592 (6), 838-851 (2018).</li><li>Vuong-Brender, T. T., Yang, X., Labouesse, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=elegans+Embryonic+Morphogenesis.">elegans Embryonic Morphogenesis.</a> Current Topics in Developmental Biology. 116, 597-616 (2016).</li><li>Wang, J. T., Seydoux, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Germ+cell+specification.">Germ cell specification.</a> Advances in Experimental Medicine and Biology. 757, 17-39 (2013).</li><li>Wang, S., 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+high-content+imaging+approach+to+profile+C.+elegans+embryonic+development.">A high-content imaging approach to profile C. elegans embryonic development.</a> Development. 146 (7), (2019).</li><li>Wang, S., Ochoa, S., Khaliullin, R. N., Gerson-Gurwitz, A., Hendel, J. M., Zhao, Z., Biggs, R., Chisholm, A. D., Desai, A., Oegema, K., Green, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Dryad Digital Repository. , (2019).</li><li>Wang, S., Ochoa, S., Khaliullin, R., Gerson-Gurwitz, A., Hendel, J., Zhao, Z., Biggs, R., Chisholm, A., Desai, A., Oegema, K., Green, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Zenodo. , (2018).</li><li>. Software to crop C. elegans embryos from multi-channel microscopy images Available from: <a target="_blank" href="https://github.com/renatkh/embryo_crop">https://github.com/renatkh/embryo_crop</a> (2018)</li><li>Schindelin, J., 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=Fiji:+an+open-source+platform+for+biological-image+analysis.">Fiji: an open-source platform for biological-image analysis.</a> Nature Methods. 9 (7), 676-682 (2012).</li><li>Schneider, C. A., Rasband, W. S., Eliceiri, K. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=NIH+Image+to+ImageJ:+25+years+of+image+analysis.">NIH Image to ImageJ: 25 years of image analysis.</a> Nature Methods. 9 (7), 671-675 (2012).</li><li>Chisholm, A. D., Hardin, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epidermal+morphogenesis.">Epidermal morphogenesis.</a> WormBook. , 1-22 (2005).</li><li>Sulston, J. E., Schierenberg, E., White, J. G., Thomson, J. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+embryonic+cell+lineage+of+the+nematode+Caenorhabditis+elegans.">The embryonic cell lineage of the nematode Caenorhabditis elegans.</a> Developmental Biology. 100 (1), 64-119 (1983).</li><li>Fakhouri, T. H., Stevenson, J., Chisholm, A. D., Mango, S. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamic+chromatin+organization+during+foregut+development+mediated+by+the+organ+selector+gene+PHA-4/FoxA.">Dynamic chromatin organization during foregut development mediated by the organ selector gene PHA-4/FoxA.</a> PLoS Genetics. 6 (8), (2010).</li><li>Zhong, 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=Genome-wide+identification+of+binding+sites+defines+distinct+functions+for+Caenorhabditis+elegans+PHA-4/FOXA+in+development+and+environmental+response.">Genome-wide identification of binding sites defines distinct functions for Caenorhabditis elegans PHA-4/FOXA in development and environmental response.</a> PLoS Genetics. 6 (2), 1000848 (2010).</li><li>Schmitz, C., Kinge, P., Hutter, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Axon+guidance+genes+identified+in+a+large-scale+RNAi+screen+using+the+RNAi-hypersensitive+Caenorhabditis+elegans+strain+nre-1(hd20)+lin-15b(hd126).">Axon guidance genes identified in a large-scale RNAi screen using the RNAi-hypersensitive Caenorhabditis elegans strain nre-1(hd20) lin-15b(hd126).</a> Proceedings of the National Academy of Sciences, USA. 104 (3), 834-839 (2007).</li><li>Canny, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+computational+approach+to+edge+detection.">A computational approach to edge detection.</a> IEEE transactions on pattern analysis and machine intelligence. 8 (6), 679-698 (1986).</li><li>Levin, M., Hashimshony, T., Wagner, F., Yanai, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Developmental+milestones+punctuate+gene+expression+in+the+Caenorhabditis+embryo.">Developmental milestones punctuate gene expression in the Caenorhabditis embryo.</a> Developmental Cell. 22 (5), 1101-1108 (2012).</li><li>Packer, J. S., 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+lineage-resolved+molecular+atlas+of+C.+elegans+embryogenesis+at+single+cell+resolution.">A lineage-resolved molecular atlas of C. elegans embryogenesis at single cell resolution.</a> BioRxiv. , (2019).</li><li>Oegema, K., Hyman, A. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+division.">Cell division.</a> WormBook. , 1-40 (2006).</li><li>Insley, P., Shaham, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Automated+C.+elegans+embryo+alignments+reveal+brain+neuropil+position+invariance+despite+lax+cell+body+placement.">Automated C. elegans embryo alignments reveal brain neuropil position invariance despite lax cell body placement.</a> PLoS One. 13 (3), 0194861 (2018).</li></ol>

This work describes a suite of tools and methods that were developed to enable larger-scale efforts to profile the function of genes in embryonic development in C. elegans. Our semi-high-throughput method allows 3D time-lapse imaging of embryonic development at 20 min resolution for 80&#8211;100 embryos in a single experiment. While this protocol can be adapted for use with any desired marker strain(s), this work demonstrates the potential of the method using two custom strains developed to monitor events during...

The C. elegans embryo is an important model system for mechanistic cell biology and analysis of cell fate specification and morphogenetic events driving embryonic development1,2,3,4,5,6,7,8,9. To date, much of the characterization of both cellular-level events and cell fate specification in the embryo has been achieved using relatively high temporal resolution one-at-a-time imaging experiments (i.e., acquisition every 10&#8211;100 s) of embryos expressing fluorescent markers. Although well suited for events on the order of seconds to tens of minutes, this approach becomes technically limiting for the characterization of longer processes, on the order of hours to days. Embryonic development from first cleavage to the end of elongation takes about 10 h. At this time-scale, semi-high-throughput methods that would allow for simultaneous lower time resolution imaging (i.e., acquisition at 5&#8211;20 min time intervals) of larger cohorts of embryos, from different conditions, would open up a new range of experiments; for example, enabling systematic large-scale screening efforts and the analysis of sufficient numbers of embryos for comparisons of the consequences of molecular perturbations.
Here, we describe a semi-high-throughput method for monitoring C. elegans embryogenesis that enables the simultaneous 3D time-lapse imaging of development in 80&#8211;100 embryos, from up to 14 different conditions, in a single overnight run. The protocol is straightforward to implement and can be carried out by any laboratory with access to a microscope with point visiting capabilities. The major steps in this protocol are outlined in Figure 1. In brief, embryos are dissected from gravid adults expressing fluorescent markers of interest and transfer young embryos (2&#8211;8 cell stage) to wells of a 384-well plate for imaging. In this format, the relatively small well size funnels embryos into a narrow area, which facilitates the identification of fields containing multiple embryos for time-lapse imaging. To maintain roughly synchronous development across the cohort of embryos, dissections are performed in chilled media and the plate is held on ice, which prevents significant development during the hour-long dissection time window. The plate is transferred to the microscope and embryos are filmed in a temperature-controlled room overnight, at 20 min time intervals, using a 60x oil immersion 1.35 NA lens, to collect the full z-range in 2 &#181;m steps. Fifty fields, each containing between 1&#8211;5 embryos, are imaged in a single overnight run. Depending on the desired experiment, the time resolution could be increased (for example, imaging at 5&#8211;10 min intervals) by proportionally decreasing the number of imaged fields.
With this protocol, even a single overnight run generates a significant amount of data (80&#8211;100 embryos spread out over 50 fields) and larger experiments can quickly become unmanageable with respect to data analysis. To facilitate processing, visualization and streamline analysis of this data, a program was built to crop out and orient embryos and perform pre-processing steps (optional), and an ImageJ macro that compiles the data to simplify viewing. These programs can be used to process images collected using conventional approaches, as they are independent of the imaging method, requiring only a single brightfield plane. The first program takes in a 4D field containing multiple embryos (GUI option or source code embryoCrop.py) or multiple 4D fields containing multiple embryos (screenCrop.py), tightly crops embryos and orients them in an anterior-posterior configuration. These programs also give users the option to perform background subtraction, drift correction, and attenuation correction. The resulting files are uniformly pre-processed, tightly cropped tiff stacks for each embryo that are amendable to automated image analysis. To make it simpler to view all embryos for each condition, an ImageJ macro (OpenandCombine_embsV2.ijm) was written, which assembles all embryos from a given condition into a single tiff stack and arrays brightfield images and maximum intensity projection color (RGB) overlays, side-by-side, for each embryo. The methods were validated by using them to characterize embryonic development after knock-down of 40 previously-described developmental genes in a pair of custom-built strains expressing fluorescent markers optimized to visualize key aspects of germ-layer specification and morphogenesis10,11. Together, the semi-high throughput embryo imaging protocol and image processing tools will enable higher sample number experiments and large-scale screening efforts aimed at understanding developmental processes. In addition, these strains will also provide an efficient means for examining the effects of molecular perturbations on embryogenesis.&#160;

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		<tr height="21">
			<td height="21" style="height:21px;width:287px;">Aspirator Tube Assembly</td>
			<td style="width:225px;">Drummond Scientific</td>
			<td style="width:491px;">2-000-000</td>
			<td style="width:85px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Calibrated Pipette (25mL)</td>
			<td>Drummond Scientific</td>
			<td>2-000-025</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Cell Voyager Software</td>
			<td>Yokogawa Electric Corp</td>
			<td>Included with CV1000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Conical Tube (15 mL )</td>
			<td>USA Scientific</td>
			<td>1475-0501</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">CV1000 Microscope</td>
			<td>Yokogawa Electric Corp</td>
			<td>CV1000</td>
			<td></td>
		</tr>
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			<td height="21" style="height:21px;">Depression slide (3-well)</td>
			<td>Erie Scientific</td>
			<td>1520-006</td>
			<td></td>
		</tr>
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			<td height="21" style="height:21px;">Dissection Microscope</td>
			<td>Nikon</td>
			<td>SMZ-645</td>
			<td></td>
		</tr>
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			<td height="21" style="height:21px;">Eppendorf Centrifuge 5810R</td>
			<td>Eppendorf</td>
			<td>5811 07336</td>
			<td></td>
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			<td height="21" style="height:21px;">ImageJ/FIJI</td>
			<td>Open Source</td>
			<td>https://imagej.net/Fiji</td>
			<td></td>
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			<td height="21" style="height:21px;">M9 Buffer</td>
			<td>Lab Prepared</td>
			<td>https://openwetware.org/wiki/M9_salts</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Microcentrifuge Tube (1.5 mLl)</td>
			<td>USA Scientific</td>
			<td>1615-5500</td>
			<td></td>
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			<td height="21" style="height:21px;">Microseal F-foil Seal</td>
			<td>Bio-Rad</td>
			<td>MSF1001</td>
			<td></td>
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			<td height="21" style="height:21px;">NGM Plates</td>
			<td>Lab Prepared</td>
			<td>http://www.wormbook.org/chapters/www_strainmaintain/strainmaintain.html#d0e214</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Scalpel #15</td>
			<td>Bard Parker</td>
			<td>REF 371615</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sensoplate Plus, 384 Well, F-bottom, Glass Bottom</td>
			<td>Greiner Bio-One</td>
			<td>781855</td>
			<td></td>
		</tr>
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			<td height="21" style="height:21px;">Tetramisole Hydrochloride</td>
			<td>Sigma Aldirch</td>
			<td>T1512-10G</td>
			<td></td>
		</tr>
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			<td height="21" style="height:21px;">Tweezers, Dumont #3</td>
			<td>Electron Microscopy Sciences</td>
			<td>0109-3-PO</td>
			<td></td>
		</tr>
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			<td height="21" style="height:21px;">U-PlanApo objective (10&#215; 0.4NA)</td>
			<td>Olympus</td>
			<td>1-U2B823</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">U-PlanApo objective (60&#215; 1.35 NA)</td>
			<td>Olympus</td>
			<td>1-U2B832</td>
			<td></td>
		</tr>
	</tbody>
</table>

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 significant challenge in characterizing the effect of molecular perturbations on C. elegans embryonic development is that it takes about 10 h for embryos to progress from first cleavage to the end of elongation at 20&#176;16. A semi-high-throughput method in which large cohorts of embryos can be simultaneously imaged is useful for events on this time-scale because it permits imaging of multiple conditions in parallel with a sufficient ensemble size for each condition to enable quantitat...

Watch this Scientific Journal Video about A Semi-high-throughput Imaging Method and Data Visualization Toolkit to Analyze C. elegans Embryonic Development at JoVE.com

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.

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 ...

C.elegans embryogenesis takes about 13 hours, and is usually monitored by filming one embryo at a time. Our protocol, however, allows the simultaneous 3D time lapse imaging of 80 to 100 embryos. The ability to collect data for large numbers of developing C.elegans embryos opens up a new range of experiments, including quantitative analysis of developmental events and large scale screens.This protocol can be easily adapted to capture different developmental processes or to image embryos expressing any combination of markers in tissues of interest. It's challenging to properly disperse the embryos within the wells. They should be close together to enable the capture of multiple embryos per field without clumping in different Z planes.We recommend using two researchers for side-by side dissection, on ice, and blocking off unused wells. We will also demonstrate some useful tricks for quickly setting up a well-staged 384-well plate. Begin by sealing a glass-bottomed 384-well plate with PCR adhesive foil to mask unused wells.Use a razor or scalpel to cut away the foil to expose a subset of the wells for use. Add 70 microliters of freshly prepared TMHC into each well and keep the plate on ice. Exclude the outer two rows of the plate to prevent edge effects.For rapid sample preparation, side-by-side dissection by two researchers is recommended. When all of the solution has been plated, use fine tweezers and a dissection microscope to transfer about 10 gravid adults into 150 microliters of ice-cold TMHC solution within one depression slide per condition. Using the tweezers and a scalpel, dissect the worms to release the embryos and load a pulled capillary pipette into a mouth aspirator.Use the mouth pipette to transfer all of the two to eight-cell stage embryos into individual wells of the prepared plate and examine the plate to confirm that no aggregates are present within any wells. When all of the embryos have been collected, settle the specimens by centrifugation and use an ethanol soaked wipe to remove any residue from the bottom of the plate. Place the plate in the plate holder of a confocal microscope equipped with a temperature-controlled environment and use the 10X objective to perform a pre-scan of each well to identify fields with suitable embryos.At the end of the scan, switch to the 60X objective, adjust the focal plane at each point to image one to four fields per well and acquire 18 Z-sections at two micrometer intervals every 20 minutes for 10 hours. When all of the images have been acquired, perform a low, whole well brightfield scan, approximately 20 to 24 hours following the start of the overnight imaging to assess the embryonic lethality. For automated cropping using embryoCropUI executable, first download the program from Zenodo and the testfiles.zip to determine whether the program is functioning properly on the platform. Once downloaded, unzip and navigate to find the embryoCropUI executable file and double click to launch the program. Select open to load the first specific 4D field of view to crop.When cropping a TIFF series with multiple dimensions load only the first image in the series within the folder. When all of the images have been loaded specify the imaging parameters, select background subtraction and attenuation correction, and set the image collection order and the microns per pixel. Then select run, a new subfolder labeled crop will be created in the same path as the uncropped folder and the cropped versions will be saved in this location.For visualization, download the open and combined MSV2 ImageJ plugin and graphic user interface instructions Zenodorepov2. docx from Zenodo and open the plugin in IMageJ. Locate lines three and four and input the location at which the image folders will be stored after the cropping.The image folder names of the conditions to be processed, and the unique alphanumeric identifier for each overnight experiment. When all of the information has been entered, click run. A window will appear that will launch a prompt to navigate to the outer folder containing the cropped image folders.Once selected another window will appear which will allow specification of the imaging parameters. Click okay. The composite file will begin to assemble.When the composite has been generated, review the files that are left open. When imaged in conjunction custom reporter strains provide informative readouts for most major developmental events. Including sulfate specification, dorsal intercalation, epidermal enclosure, elongation, and neurogenesis.24 hours after injection is an optimal time point for imaging. As of this time maternal protein depletion and inhibition of zygotic gene expression are both effective yielding distinct, highly reproducible signature phenotypes across a broad spectrum of genes involved in sulfate specification and morphogenesis. It is important to be patient during the dissection and embryo selection steps.Minimizing the clumping and selecting the appropriate focal planes are also essential to ensuring high quality data. Our protocol can be performed with a variety of fluorescent marker strains and in combination with RNAi or mutants. Our processing tools allow for streamlined, automated, or manual data analysis.Using the strains, methods, and tools described here our group performed an RNAi-based high content screen targeting approximately 2, 000 genes required for embryonic development. Semi-high throughput methods were essential for this project.

a-semi-high-throughput-imaging-method-data-visualization-toolkit-to

Research

JoVE Journal

Developmental Biology

Visualization of Chondrocyte Intercalation and Directional Proliferation via Zebrabow Clonal Cell Analysis in the Embryonic Meckel&#8217;s Cartilage

Development of the vertebrate craniofacial structures requires precise coordination of cell migration, proliferation, adhesion and differentiation. Patterning of the Meckel&#39;s cartilage, a first pharyngeal arch derivative, involves the migration of cranial neural crest (CNC) cells and the progressive partitioning, proliferation and organization of differentiated chondrocytes. Several studies have described CNC migration during lower jaw&#160;morphogenesis, but the details of how the chondrocytes achieve organization in the growth and extension of Meckel&#8217;s cartilage remains unclear. The sox10 restricted and chemically induced Cre recombinase-mediated recombination generates permutations of distinct fluorescent proteins (RFP, YFP and CFP), thereby creating a multi-spectral labeling of progenitor cells and their progeny, reflecting distinct clonal populations. Using confocal time-lapse photography, it is possible to observe the chondrocytes behavior during the development of the zebrafish Meckel&#8217;s cartilage.
Multispectral cell labeling enables scientists to demonstrate extension of the Meckel&#8217;s chondrocytes.&#160;During extension phase of the Meckel&#8217;s cartilage, which prefigures the mandible, chondrocytes intercalate to effect extension as they stack in an organized single-cell layered row. Failure of this organized intercalating process to mediate cell extension provides the cellular mechanistic explanation for hypoplastic mandible that we observe in mandibular malformations.

Visualization of Chondrocyte Intercalation and Directional Proliferation via Zebrabow Clonal Cell Analysis in the Embryonic Meckel&amp;#8217;s Cartilage

Cell organization of craniofacial bones has long been hypothesized but never directly visualized. Multi-spectral cell labeling and in vivo live imaging allows visualization of dynamic cell behavior in zebrafish lower jaw. Here, we detail the protocol to manipulate Zebrabow transgenic fish and directly observe cell intercalation and morphological changes of chondrocytes in the Meckel&#8217;s cartilage.

Development of the vertebrate craniofacial structures requires precise coordination of cell migration, proliferation, adhesion and differentiation. ...

Transcriptome Profiling of In-Vivo Produced Bovine Pre-implantation Embryos Using Two-color Microarray Platform

Early embryonic loss is a large contributor to infertility in cattle. Moreover, bovine becomes an interesting model to study human preimplantation embryo development due to their similar developmental process. Although genetic factors are known to affect early embryonic development, the discovery of such factors has been a serious challenge. Microarray technology allows quantitative measurement and gene expression profiling of transcript levels on a genome-wide basis. One of the main decisions that have to be made when planning a microarray experiment is whether to use a one- or two-color approach. Two-color design increases technical replication, minimizes variability, improves sensitivity and accuracy as well as allows having loop designs, defining the common reference samples. Although microarray is a powerful biological tool, there are potential pitfalls that can attenuate its power. Hence, in this technical paper we demonstrate an optimized protocol for RNA extraction, amplification, labeling, hybridization of the labeled amplified RNA to the array, array scanning and data analysis using the two-color analysis strategy.

Transcriptome Profiling of In-Vivo Produced Bovine Pre-implantation Embryos Using Two-color Microarray Platform

Microarray technology allows quantitative measurement and gene expression profiling of transcripts on a genome-wide basis. Therefore, this protocol provides an optimized technical procedure in a two-color custom made bovine array using Day 7 bovine embryos to demonstrate the feasibility of using low amount of total RNA.

Early embryonic loss is a large contributor to infertility in cattle. Moreover, bovine becomes an interesting model to study human preimplantation embryo ...

A Versatile Mounting Method for Long Term Imaging of Zebrafish Development

Zebrafish embryos offer an ideal experimental system to study complex morphogenetic processes due to their ease of accessibility and optical transparency. In particular, posterior body elongation is an essential process in embryonic development by which multiple tissue deformations act together to direct the formation of a large part of the body axis. In order to observe this process by long-term time-lapse imaging it is necessary to utilize a mounting technique that allows sufficient support to maintain samples in the correct orientation during transfer to the microscope and acquisition. In addition, the mounting must also provide sufficient freedom of movement for the outgrowth of the posterior body region without affecting its normal development. Finally, there must be a certain degree in versatility of the mounting method to allow imaging on diverse imaging set-ups. Here, we present a mounting technique for imaging the development of posterior body elongation in the zebrafish D. rerio. This technique involves mounting embryos such that the head and yolk sac regions are almost entirely included in agarose, while leaving out the posterior body region to elongate and develop normally. We will show how this can be adapted for upright, inverted and vertical light-sheet microscopy set-ups. While this protocol focuses on mounting embryos for imaging for the posterior body, it could easily be adapted for the live imaging of multiple aspects of zebrafish development.

Here, we present a versatile mounting method that allows for the long-term time-lapse imaging of the posterior body development of live zebrafish embryos without perturbing normal development.

Zebrafish embryos offer an ideal experimental system to study complex morphogenetic processes due to their ease of accessibility and optical transparency. ...

A Simple Method to Identify Kinases That Regulate Embryonic Stem Cell Pluripotency by High-throughput Inhibitor Screening

Embryonic stem cells (ESCs) can self-renew or differentiate into all cell types, a phenomenon known as pluripotency. Distinct pluripotent states have been described, termed &#34;na&#239;ve&#34; and &#34;primed&#34; pluripotency. The mechanisms that control na&#239;ve-primed transition are poorly understood. In particular, we remain poorly informed about protein kinases that specify na&#239;ve and primed pluripotent states, despite increasing availability of high-quality tool compounds to probe kinase function. Here, we describe a scalable platform to perform targeted small molecule screens for kinase regulators of the na&#239;ve-primed pluripotent transition in mouse ESCs. This approach utilizes simple cell culture conditions and standard reagents, materials and equipment to uncover and validate kinase inhibitors with hitherto unappreciated effects on pluripotency. We discuss potential applications for this technology, including screening of other small molecule collections such as increasingly sophisticated kinase inhibitors and emerging libraries of epigenetic tool compounds.

Here, we present a quantitative and scalable protocol to perform targeted small molecule screens for kinase regulators of the na&#239;ve-primed pluripotent transition.

Embryonic stem cells (ESCs) can self-renew or differentiate into all cell types, a phenomenon known as pluripotency. Distinct pluripotent states have been ...

Visualization and Quantitative Analysis of Embryonic Angiogenesis in Xenopus tropicalis

Blood vessels supply oxygen and nutrients throughout the body, and the formation of the vascular network is under tight developmental control. The efficient in vivo visualization of blood vessels and the reliable quantification of their complexity are key to understanding the biology and disease of the vascular network. Here, we provide a detailed method to visualize blood vessels with a commercially available fluorescent dye, human plasma acetylated low density lipoprotein DiI complex (DiI-AcLDL), and to quantify their complexity in Xenopus tropicalis. Blood vessels can be labeled by a simple injection of DiI-AcLDL into the beating heart of an embryo, and blood vessels in the entire embryo can be imaged in live or fixed embryos. Combined with gene perturbation by the targeted microinjection of nucleic acids and/or the bath application of pharmacological reagents, the roles of a gene or of a signaling pathway on vascular development can be investigated within one week without resorting to sophisticated genetically engineered animals. Because of the well-defined venous system of Xenopus and its stereotypic angiogenesis, the sprouting of pre-existing vessels, vessel complexity can be quantified efficiently after perturbation experiments. This relatively simple protocol should serve as an easily accessible tool in diverse fields of cardiovascular research.

Visualization and Quantitative Analysis of Embryonic Angiogenesis in Xenopus tropicalis

This protocol demonstrates a fluorescence-based method to visualize the vasculature and to quantify its complexity in Xenopus tropicalis. Blood vessels can be imaged minutes after the injection of a fluorescent dye into the beating heart of an embryo after genetic and/or pharmacological manipulations to study cardiovascular development in vivo.

Blood vessels supply oxygen and nutrients throughout the body, and the formation of the vascular network is under tight developmental control. The ...

Multi-Photon Time Lapse Imaging to Visualize Development in Real-time: Visualization of Migrating Neural Crest Cells in Zebrafish Embryos

Congenital eye and craniofacial anomalies reflect disruptions in the neural crest, a transient population of migratory stem cells that give rise to numerous cell types throughout the body. Understanding the biology of the neural crest has been limited, reflecting a lack of genetically tractable models that can be studied in vivo and in real-time. Zebrafish is a particularly important developmental model for studying migratory cell populations, such as the neural crest. To examine neural crest migration into the developing eye, a combination of the advanced optical techniques of laser scanning microscopy with long wavelength multi-photon fluorescence excitation was implemented to capture high-resolution, three-dimensional, real-time videos of the developing eye in transgenic zebrafish embryos, namely Tg(sox10:EGFP) and Tg(foxd3:GFP), as sox10 and foxd3 have been shown in numerous animal models to regulate early neural crest differentiation and likely represent markers for neural crest cells. Multi-photon time-lapse imaging was used to discern the behavior and migratory patterns of two neural crest cell populations contributing to early eye development. This protocol provides information for generating time-lapse videos during zebrafish neural crest migration, as an example, and can be further applied to visualize the early development of many structures in the zebrafish and other model organisms.

A combination of the advanced optical techniques of laser scanning microscopy with long wavelength multi-photon fluorescence excitation was implemented to capture high-resolution, three-dimensional, real-time imaging of neural crest migration in Tg(sox10:EGFP) and Tg(foxd3:GFP) zebrafish embryos.

Congenital eye and craniofacial anomalies reflect disruptions in the neural crest, a transient population of migratory stem cells that give rise to ...

Immobilization of Caenorhabditis elegans to Analyze Intracellular Transport in Neurons

Axonal transport and intraflagellar transport (IFT) are essential for axon and cilia morphogenesis and function. Kinesin superfamily proteins and dynein are molecular motors that regulate anterograde and retrograde transport, respectively. These motors use microtubule networks as rails. Caenorhabditis elegans (C. elegans) is a powerful model organism to study axonal transport and IFT in vivo. Here, I describe a protocol to observe axonal transport and IFT in living C. elegans. Transported cargo can be visualized by tagging cargo proteins using fluorescent proteins such as green fluorescent protein (GFP). C. elegans is transparent and GFP-tagged cargo proteins can be expressed in specific cells under cell-specific promoters. Living worms can be fixed by microbeads on 10% agarose gel without killing or anesthetizing the worms. Under these conditions, cargo movement can be directly observed in the axons and cilia of living C. elegans without dissection. This method can be applied to the observation of any cargo molecule in any cells by modifying the target proteins and/or the cells they are expressed in. Most basic proteins such as molecular motors and adaptor proteins that are involved in axonal transport and IFT are conserved in C. elegans. Compared to other model organisms, mutants can be obtained and maintained more easily in C. elegans. Combining this method with various C. elegans mutants can clarify the molecular mechanisms of axonal transport and IFT.

Immobilization of Caenorhabditis elegans to Analyze Intracellular Transport in Neurons

Caenorhabditis elegans (C. elegans) is a good model to study axonal and intracellular transport. Here, I describe a protocol for in vivo recording and analysis of axonal and intraflagellar transport in C. elegans.

Axonal transport and intraflagellar transport (IFT) are essential for axon and cilia morphogenesis and function. Kinesin superfamily proteins and dynein ...

Organotypic Culture Method to Study the Development Of Embryonic Chicken Tissues

The embryonic chicken is commonly used as a reliable model organism for vertebrate development. Its accessibility and short incubation period makes it ideal for experimentation. Currently, the study of these developmental pathways in the chicken embryo is conducted by applying inhibitors and drugs at localized sites and at low concentrations using a variety of methods. In vitro tissue&#160;culturing is a technique that enables the study of tissues separated from the host organism, while simultaneously bypassing many of the physical limitations present when working with whole embryos, such as the susceptibility of embryos to high doses of potentially lethal chemicals. Here, we present an organotypic culturing protocol for culturing the embryonic chicken half head in vitro, which presents new opportunities for the examination of developmental processes beyond the currently established methods.

Here, we present an organotypic culturing protocol to grow embryonic chicken organs in vitro. Using this method, the development of embryonic chicken tissue can be studied, while maintaining a high degree of control over the culture environment.

The embryonic chicken is commonly used as a reliable model organism for vertebrate development. Its accessibility and short incubation period makes it ...

Computational Analysis of the Caenorhabditis elegans Germline to Study the Distribution of Nuclei, Proteins, and the Cytoskeleton

The Caenorhabditis elegans (C. elegans) germline is used to study several biologically important processes including stem cell development, apoptosis, and chromosome dynamics. While the germline is an excellent model, the analysis is often two dimensional due to the time and labor required for three-dimensional analysis. Major readouts in such studies are the number/position of nuclei and protein distribution within the germline. Here, we present a method to perform automated analysis of the germline using confocal microscopy and computational approaches to determine the number and position of nuclei in each region of the germline. Our method also analyzes germline protein distribution that enables the three-dimensional examination of protein expression in different genetic backgrounds. Further, our study shows variations in cytoskeletal architecture in distinct regions of the germline that may accommodate specific spatial developmental requirements. Finally, our method enables automated counting of the sperm in the spermatheca of each germline. Taken together, our method enables rapid and reproducible phenotypic analysis of the C. elegans germline.

Computational Analysis of the Caenorhabditis elegans Germline to Study the Distribution of Nuclei, Proteins, and the Cytoskeleton

We present an automated method for three-dimensional reconstruction of the Caenorhabditis elegans germline. Our method determines the number and position of each nucleus within the germline and analyses germline protein distribution and cytoskeletal structure.

The Caenorhabditis elegans (C. elegans) germline is used to study several biologically important processes including stem cell development, apoptosis, and ...

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.

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