We thank Dr. Gaudenz Danuser and members of his lab for creating the plusTipTracker software and for helpful discussion regarding using the software, in particular Maria Bagonis and Sebastien Besson. We especially thank the Boston College Media Center for their assistance and support in the creation and editing of the video. We also thank members of the Lowery Lab for useful discussions and constructive criticism, and Abigail Antoine for proof-reading the manuscript. This work was funded by an NIH R00 MH095768 award to LAL.

This protocol and video are meant to serve as a companion to the original paper describing the software package in more detail4, as well as the Technical Report that comes with the software download on the Danuser Lab website. Readers are encouraged to review these documents carefully if there are additional questions regarding using the software.
1. Prior to Image Analysis
<ol>
	<li>Convert each time-lapse movie into a sequence of TIFF (Tagged Image File Format) image files. If there are multiple growth cones/cells in a given movie, first crop each growth cone/cell to create its own image sequence. 
	Note: This is not necessary, as individual regions-of-interest (ROI) can be selected within plusTipTracker. However, using smaller image dimensions increases the speed of the computational processing, so this step is recommended if there is significant blank space in the image.</li>
	<li>Save each TIFF series in its own folder called &#8220;images&#8221; within a path that MATLAB&#160;is set to access (note that &#8220;images&#8221; is case-sensitive). To add a new path, navigate to the relevant file directory in the &#8220;Current Folder&#8221; window, right-click on the directory icon, and select &#8220;Add to Path &#8211; Selected Folders and Sub-Folders&#8221;. It is important that the plusTipTracker software folder be added to the Path, as well.</li>
</ol>
2. plusTipGetTracks
Note: The first step in image analysis is to detect the EB1-GFP comets, link the comets into tracks, and determine the parameters of microtubule dynamics. This is obtained with the command &#8220;plusTipGetTracks&#8221;4.
<ol>
	<li>To begin analysis, open MATLAB&#160;application and type &#8220;plusTipGetTracks&#8221; into the command window. This will cause a new dialog box to appear.</li>
	<li>Click on &#8220;Set Up New Projects&#8221; and select one (or more) of the previous TIFF image series by selecting the appropriate &#8220;images&#8221; folder (or directories containing &#8220;images&#8221; folders). Upon completion of this step, a file directory (roi_1) will be created (in the same folder that holds &#8220;images&#8221;) that will contain the future data files. Note: the &#8220;Set Up New Projects&#8221; step can be completed ahead of time, during a separate session.</li>
	<li>A new window will appear: &#8220;Select a polygon, right-click on last point, and click on &#8216;Create Mask&#8217;&#8221;. Click on &#8220;OK&#8221;. The first image of the selected image series will then be displayed. Use the mouse to click and create a polygon that encompasses the entirety of the growth cone. Double click the mouse to close the polygon.</li>
	<li>Once the polygon has been closed, a dialog box will appear: &#8220;Do you want to select another ROI?&#8221; If the image has another growth cone to analyze, select &#8220;Yes&#8221;; otherwise select &#8220;No&#8221;.</li>
	<li>Select the projects that will be immediately analyzed. Click on &#8220;Select Projects&#8221; and select the folder (roi_X) to analyze.</li>
	<li>A listSelectGUI screen will appear. Select the project(s) from the left side of the screen and move them over to the right side of the screen. Click on &#8220;OK&#8221;. Choose a location to save the project list and click on &#8220;Save&#8221;.</li>
	<li>Select &#8220;Detection&#8221;, &#8220;Tracking&#8221;, and &#8220;Post-Processing&#8221;. Once these selections have been made, the right side of the dialog box will become configurable. Configure each option.
	<ol>
		<li>These parameters are used to link detected comets into MT tracks. Details for choosing these control parameters for tracking are included on pages 9-10 of the Technical Report PDF which accompanies the software package download; read this report carefully if problems are encountered. For the purposes of tracking EB1-GFP comets in Xenopus laevis growth cones, use the following Tracking Settings: Search Radius Range (pixels) 5-12, Minimum Sub-Track Length (frames) 3; Max Gap Length (frames) 8; Max Shrinkage Factor 0.8, Max angle Forward 50, Max angle Backward 10, Fluctuation Radius 2.5. These settings are shown in Figure 1.</li>
	</ol>
	 
	Note: Max Shrinkage Factor is set to reduce the number of &#8220;backward gaps&#8221; detected, as &#8220;backward gaps&#8221; are not useful to analyze in the context of growth cones, given the crowded conditions and likely errors in track linkages. Additionally, both Max Forward angle as well as Fluctuation Radius are set relatively high, as growth cone MTs exhibit small frequent translocations in addition to growths and shrinkages, and increasing these control settings allows for this increased movement during the linkage step.
	<ol>
		<li>Fill in the Post-Processing Settings depending upon the desired specific image acquisition settings.</li>
	</ol>
	</li>
	<li>Once the settings have been configured, click on &#8220;Start&#8221;. The software will run whichever settings have been selected. This may take minutes to hours, depending on the number of projects selected and their sizes. The Command Window displays the estimated time remaining for each function. When the plusTipGetTracks step is completed, the Command Window will display &#8220;Finished!&#8221; 
	Note: A long list of specific parameters of MT dynamics have now been calculated by this software (see Reference 4 for details), but for analysis of MT dynamics in growth cones, the most useful parameters to examine are MT growth track velocity (in microns/minute), growth track lifetime (in seconds), and growth track length (in microns).</li>
</ol>
3. plusTipSeeTracks
Note: Now that the microtubule tracks have been defined, the function &#8220;plustipSeeTracks&#8221; is used for track visualization4. This function can provide multiple outputs for visualization, including spatial MT dynamics maps and speed movies, but here, the focus is solely on using &#8220;Track Movies&#8221; to display MT tracks superimposed on the growth cone images. While plusTipGetTracks can analyze multiple movies at a time, plusTipSeeTracks can only analyze one movie at a time.
<ol>
	<li>Type &#8220;plusTipSeeTracks&#8221; into the command window.</li>
	<li>After the dialog box loads, click on &#8220;Select Project&#8221;. Select the parent directory containing the project to visualize and click on &#8220;Select Folder&#8221;. A new window will appear: &#8220;Select the project&#160;you want to visualize&#8221;. Choose the file to visualize and click on &#8220;OK&#8221;.</li>
	<li>Next, click on &#8220;Select Saved ROI&#8221;. Navigate to the same roi_X folder as the one selected in the previous step and select the file named &#8220;roiYX&#8221;.</li>
	<li>Click on &#8220;Select Output Directory&#8221; to designate where MATLAB&#160;will save the track visualization files. Note: We recommend using the same folder that contains the rest of the data.</li>
	<li>Select &#8220;Make Track Movie&#8221; and a screen will appear displaying all of the tracks plusTipGetTracks calculated from the +TIP comets. This step saves the tracked time series in a movie format, in the file &#8220;allTracks_X_X_X&#8221;. There is an option for saving the movie as an AVI, otherwise the default format is as a Quicktime.mov file.</li>
</ol>

4. plusTipGroupAnalysis
Note: This final function is used to create groups of movies for analysis and comparison of their MT track parameters.
<ol>
	<li>Type &#8220;plusTipGroupAnalysis&#8221; into the command window. To manually select the groups to compare, first de-select &#8220;Auto group from hierarchy&#8221;. Then, click on &#8220;Select projects&#8221;. Navigate to the parent directories containing all the roi_X folders to analyze.</li>
	<li>A listSelectGUI screen will appear. Select all of the projects to include in the groups from the left side of the screen and move them over to the right side of the screen. Click on &#8220;OK&#8221;. Choose a location to save the project list and click on &#8220;Save&#8221;.</li>
	<li>A window will appear: &#8220;Please select first group from the list&#8221;. Click on &#8220;OK&#8221;. The listSelectGUI window will display again. This time, select only those files that correspond to the first group that should be pooled together. Click on &#8220;OK&#8221;.</li>
	<li>Then, enter the group name, and click on &#8220;OK&#8221;. A window will appear: &#8220;Select another group?&#8221; Answer accordingly and continue selecting groups. A window will appear: &#8220;Select a location to save your group list&#8221;. Navigate to the location and click on &#8220;Save&#8221;.</li>
	<li>Click on &#8220;Select Output Directory&#8221; to choose where the output folders will be stored.</li>
	<li>Select which type of group analysis to conduct &#8211; whether the MT tracks should be pooled for each group or per cell analysis should be performed. The recommended statistical tests are already designated. To include all of the tracks in the analysis, de-select &#8220;Remove tracks at beginning/end of movie&#8221;. Otherwise, having this box selected removes any MT growth tracks that are in process as the movie begins or ends.</li>
	<li>After the Group Analysis selection is made, select &#8220;Compare groups&#8221;.</li>
</ol>

<ol>
	<li>Stepanova, T., 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=Visualization+of+microtubule+growth+in+cultured+neurons+via+the+use+of+EB3-GFP+(end-binding+protein+3-green+fluorescent+protein).">Visualization of microtubule growth in cultured neurons via the use of EB3-GFP (end-binding protein 3-green fluorescent protein).</a> The Journal of neuroscience : the official journal of the Society for Neuroscience. 23, 2655-2664 (2003).</li><li>Lee, 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=The+microtubule+plus+end+tracking+protein+Orbit/MAST/CLASP+acts+downstream+of+the+tyrosine+kinase+Abl+in+mediating+axon+guidance.">The microtubule plus end tracking protein Orbit/MAST/CLASP acts downstream of the tyrosine kinase Abl in mediating axon guidance.</a> Neuron. , 913-926 (2004).</li><li>Purro, S. A., 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=Wnt+regulates+axon+behavior+through+changes+in+microtubule+growth+directionality:+a+new+role+for+adenomatous+polyposis+coli.">Wnt regulates axon behavior through changes in microtubule growth directionality: a new role for adenomatous polyposis coli.</a> The Journal of neuroscience : the official journal of the Society for Neuroscience. 28, 8644-8654 (2008).</li><li>Applegate, K. T., 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=plusTipTracker:+Quantitative+image+analysis+software+for+the+measurement+of+microtubule+dynamics.">plusTipTracker: Quantitative image analysis software for the measurement of microtubule dynamics.</a> Journal of structural biology. 176, 168-184 (2011).</li><li>Lowery, L. A., Faris, A. E., Stout, A., Van Vactor, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neural+Explant+Cultures+from+Xenopus+laevis.">Neural Explant Cultures from Xenopus laevis.</a> Journal of visualized experiments : JoVE. (68), e4232 (2012).</li><li>Long, J. B., 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=Multiparametric+analysis+of+CLASP-interacting+protein+functions+during+interphase+microtubule+dynamics.">Multiparametric analysis of CLASP-interacting protein functions during interphase microtubule dynamics.</a> Molecular and cellular biology. 33, 1528-1545 (2013).</li><li>Myers, K. A., Applegate, K. T., Danuser, G., Fischer, R. S., Waterman, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Distinct+ECM+mechanosensing+pathways+regulate+microtubule+dynamics+to+control+endothelial+cell+branching+morphogenesis.">Distinct ECM mechanosensing pathways regulate microtubule dynamics to control endothelial cell branching morphogenesis.</a> The Journal of cell biology. 192, 321-334 (2011).</li><li>Nishimura, Y., Applegate, K., Davidson, M. W., Danuser, G., Waterman, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Automated+screening+of+microtubule+growth+dynamics+identifies+MARK2+as+a+regulator+of+leading+edge+microtubules+downstream+of+Rac1+in+migrating+cells.">Automated screening of microtubule growth dynamics identifies MARK2 as a regulator of leading edge microtubules downstream of Rac1 in migrating cells.</a> PLoS One. 7, e41413 (2012).</li><li>Akhmanova, A., Steinmetz, M. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tracking+the+ends:+a+dynamic+protein+network+controls+the+fate+of+microtubule+tips.">Tracking the ends: a dynamic protein network controls the fate of microtubule tips.</a> Nature reviews. Molecular cell biology. 9, 309-322 (2008).</li><li>Schuyler, S. C., Pellman, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microtubule+'plus-end-tracking+proteins':+The+end+is+just+the+beginning.">Microtubule 'plus-end-tracking proteins': The end is just the beginning.</a> Cell. 105, 421-424 (2001).</li><li>Mitchison, T., Kirschner, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamic+instability+of+microtubule+growth.">Dynamic instability of microtubule growth.</a> Nature. 312, 237-242 (1984).</li><li>Mimori-Kiyosue, Y., Shiina, N., Tsukita, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+dynamic+behavior+of+the+APC-binding+protein+EB1+on+the+distal+ends+of+microtubules.">The dynamic behavior of the APC-binding protein EB1 on the distal ends of microtubules.</a> Current biology : CB. 10, 865-868 (2000).</li><li>Dixit, R., 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=Microtubule+plus-end+tracking+by+CLIP-170+requires+EB1.">Microtubule plus-end tracking by CLIP-170 requires EB1.</a> Proceedings of the National Academy of Sciences of the United States of America. 106, 492-497 (2009).</li><li>Li, W., 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=EB1+promotes+microtubule+dynamics+by+recruiting+Sentin+in+Drosophila+cells.">EB1 promotes microtubule dynamics by recruiting Sentin in Drosophila cells.</a> The Journal of cell biology. 193, 973-983 (2011).</li><li>Maurer, S. P., 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=EB1+accelerates+two+conformational+transitions+important+for+microtubule+maturation+and+dynamics.">EB1 accelerates two conformational transitions important for microtubule maturation and dynamics.</a> Current biology : CB. 24, 372-384 (2014).</li><li>Zanic, M., Widlund, P. O., Hyman, A. A., Howard, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synergy+between+XMAP215+and+EB1+increases+microtubule+growth+rates+to+physiological+levels.">Synergy between XMAP215 and EB1 increases microtubule growth rates to physiological levels.</a> Nature cell biology. 15, 688-693 (2013).</li><li>Lowery, L. 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The authors declare that they have no competing financial interests.

PlusTipTracker provides a straightforward, graphical user interface to quickly and automatically detect virtually all visible EB1-GFP comets in a cell or growth cone, link the comets into tracks, and calculate MT parameters. Other publications have reported the design of similar types of software (for example, Marx et al. also utilized quantitative analysis of tagged EB1 dynamics in growth cones18). But, this software appears to be unique in its ease of access, as it is freely downloadable from the we...

The goal of this method is to obtain quantitative information regarding microtubule (MT) plus-end-tracking protein (+TIP) dynamics in living growth cones. MT +TIPs are a group of proteins that localize to the plus-ends of MTs9,10. They perform a range of functions to regulate parameters of MT dynamic instability11, including rates of polymerization, catastrophe, and rescue. One well-used method for analyzing MT dynamics is to track the behavior of the +TIP EB1, which binds specifically to growing MT plus-ends1,12. EB1 is known to recruit several other proteins to growing MT plus-ends13,14, and has recently been established as a MT maturation factor15, promoting both MT growth and catastrophe frequency15,16.
Many studies of MT dynamics within growth cones have utilized hand-tracking methods to measure changes in EB1-GFP dynamics over time1-3, as EB1 localization to MT plus-ends can be used as a marker for MT polymerization. A key benefit for examining EB1-GFP comets as a proxy for MT growth is that MT dynamics can be measured even in regions of significant MT overlap. While the method of hand-tracking EB1-GFP comets has provided useful insights into MT behaviors1-3, it is time-consuming and can be biased. Additionally, as aberrant growth cone behaviors are likely the result of minute shifts in cytoskeletal dynamics, analyzing only a small subset of MTs (usually necessary when hand-tracking) may miss significant information.
Thus, we measure global MT dynamics parameters using the software package, plusTipTracker4, after the acquisition of high-resolution, live images of tagged EB1 in cultured embryonic growth cones5. This software, developed in the Danuser Lab, has been used in several studies characterizing MT dynamics in various cell types6-8. It is an open-source, user-friendly, MATLAB-based package that includes automated detection, tracking, visualization, and analysis for movies of fluorescently-labeled +TIPs. A long list of specific parameters of MT dynamics are calculated by this software (see Reference 4 for details), but for analysis of MT dynamics in growth cones, the most useful parameters are MT growth track velocity (in microns/minute), growth track lifetime (in seconds), and growth track length (in microns). The software can be downloaded directly from the Danuser Lab website (under &#8220;Software&#8221;). While the Danuser Lab currently supports a newer interface for +TIP tracking analysis, which is incorporated into a software package called u-track 2.0, the original, stand-alone software will remain available. The underlying algorithms between the two programs are the same (at least as of 2014), with only a difference of interface and analysis outputs. For the novice user with little MATLAB&#160;and/or computational analysis experience, plusTipTracker has more user-friendly features, including automated statistical parameter outputs.
Here, we describe the steps for analyzing images of EB1-GFP dynamics in cultured Xenopus laevis growth cones. This protocol was utilized in a recent paper examining MT dynamics17. See also Lowery et al. 20125 for detailed instructions regarding culturing growth cones expressing EB1-GFP. While this paper primarily focused on examining EB1-GFP dynamics in growth cones, the same protocol can be used for other cell types17. For all cell types, the time interval between frames should be between 0.5-2 sec&#160;for optimal +TIP tracking. A time interval of up to 4 seconds between frames is possible, but this increased interval time results in additional tracking errors.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">plusTipTracker software</td>
			<td style="width:116px;">Danuser Lab</td>
			<td style="width:345px;"><a href="http://lccb.hms.harvard.edu/software.html">http://lccb.hms.harvard.edu/software.html</a></td>
			<td style="width:172px;">This software may be hosted by another website in the future.&#160; If the listed site does not exist, search &#34;Danuser Lab Software&#34; on a web search engine to find the site.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">Matlab software</td>
			<td style="width:116px;">Mathworks</td>
			<td><a href="http://www.mathworks.com/products/matlab/">http://www.mathworks.com/products/matlab/</a></td>
			<td></td>
		</tr>
	</tbody>
</table>

using plustiptracker software to measure microtubule dynamics in xenopus laevis growth cones

Microtubule (MT) plus-end-tracking proteins (+TIPs) localize to the growing plus-ends of MTs and regulate MT dynamics1,2. One of the most well-known and widely-utilized +TIPs for analyzing MT dynamics is the End-Binding protein, EB1, which binds all growing MT plus-ends, and thus, is a marker for MT polymerization1. Many studies of EB1 behavior within growth cones have used time-consuming and biased computer-assisted, hand-tracking methods to analyze individual MTs1-3. Our approach is to quantify global parameters of MT dynamics using the software package, plusTipTracker4, following the acquisition of high-resolution, live images of tagged EB1 in cultured embryonic growth cones5. This software is a MATLAB-based, open-source, user-friendly package that combines automated detection, tracking, visualization, and analysis for movies of fluorescently-labeled +TIPs. Here, we present the protocol for using plusTipTracker for the analysis of fluorescently-labeled +TIP comets in cultured Xenopus laevis growth cones. However, this software can also be used to characterize MT dynamics in various cell types6-8.

Using this software as described here will provide several files of information that quantify +TIP dynamics in living cells.
The function plusTipGetTracks identifies the tracks (using example settings shown in Figure 1), and then provides parameters regarding the +TIP tracks. To view the information that the software has obtained, go into the roi_X directory that was created in step 2.2. The &#8220;feat&#8221; folder contains &#8220;overlayImages&#8221;, which is a series of i...

Watch this Scientific Journal Video about Using plusTipTracker Software to Measure Microtubule Dynamics in Xenopus laevis Growth Cones at JoVE.com

Using plusTipTracker Software to Measure Microtubule Dynamics in Xenopus laevis Growth Cones

The MATLAB-based, open source software package, plusTipTracker, can be used to analyze image series of fluorescently-labeled +TIPs to quantify microtubule dynamics.

Using plusTipTracker Software to Measure Microtubule Dynamics in Xenopus laevis Growth Cones

Microtubule (MT) plus-end-tracking proteins (+TIPs) localize to the growing plus-ends of MTs and regulate MT dynamics1,2. One of the most well-known and ...

using-plustiptracker-software-to-measure-microtubule-dynamics-xenopus

Research

JoVE Journal

Biology

10.3K Views.  Boston College.  Il pacchetto software basato su MATLAB open source, plusTipTracker, può essere utilizzato per analizzare serie di immagini di + TIP fluorescenza marcata per quantificare la dinamica dei microtubuli. 

Video: Using plusTipTracker Software to Measure Microtubule Dynamics in Xenopus laevis Growth Cones

Plastic Embedding and Sectioning of Xenopus laevis Embryos

Plastic sections maintain true tissue morphology in thin sections of tissue that can be immunostained with fluorescent secondary antibodies, making this method more useful than paraffin-embedded or frozen sections for many types of tissue. The method for staining, plastic embedding, and sectioning is demonstrated in this video.

Neuronal Cell Cultures from Aplysia for High-Resolution Imaging of Growth Cones

Neuronal growth cones are the highly motile structures at the tip of axons that can detect guidance cues in the environment and transduce this information into directional movement towards the appropriate target cell. To fully understand how guidance information is transmitted from the cell surface to the underlying dynamic cytoskeletal networks, one needs a model system suitable for live cell imaging of protein dynamics at high temporal and spatial resolution. Typical vertebrate growth cones are too small to quantitatively analyze F-actin and microtubule dynamics. Neurons from the sea hare Aplysia californica are 5-10 times larger than vertebrate neurons, can easily be kept at room temperature and are very robust cells for micromanipulation and biophysical measurements. Their growth cones have very defined cytoplasmic regions and a well-described cytoskeletal system. The neuronal cell bodies can be microinjected with a variety of probes for studying growth cone motility and guidance. In the present protocol we demonstrate a procedure for dissection of the abdominal ganglion, culture of bag cell neurons and setting up an imaging chamber for live cell imaging of growth cones.

Aplysia californica neurons develop large growth cones in culture that are excellent for high-resolution imaging of growth cone motility and guidance. Here, we present a protocol for dissection and plating of Aplysia bag cell neurons as well as for setting up a chamber for live cell imaging.

Neuronal growth cones are the highly motile structures at the tip of axons that can detect guidance cues in the environment and transduce this information ...

Obtaining Eggs from Xenopus laevis Females

The eggs of Xenopus laevis intact, lysed, and/or fractionated are useful for a wide variety of experiments. This protocol shows how to induce egg laying, collect and dejelly the eggs, and sort the eggs to remove any damaged eggs.

The eggs of Xenopus laevis intact, lysed, and/or fractionated are useful for a wide variety of experiments. This protocol shows how to induce egg laying, collect and dejelly the eggs, and sort the eggs to remove any damaged eggs.

The eggs of Xenopus laevis intact, lysed, and/or fractionated are useful for a wide variety of experiments. This protocol shows how to induce egg laying, ...

Preparation and Fractionation of Xenopus laevis Egg Extracts

Crude and fractionated Xenopus egg extracts can be used to provide ingredients for reconstituting cellular processes for morphological and biochemical analysis. Egg lysis and differential centrifugation are used to prepare the crude extract which in turn in used to prepare fractionated extracts and light membrane preparations.

Crude and fractionated Xenopus egg extracts can be used to provide ingredients for reconstituting cellular processes for morphological and biochemical analysis. Egg lysis and differential centrifugation are used to prepare the crude extract which in turn in used to prepare fractionated extracts and light membrane preparations.

Crude and fractionated Xenopus egg extracts can be used to provide ingredients for reconstituting cellular processes for morphological and biochemical ...

Microinjection of Xenopus Laevis Oocytes

Microinjection of Xenopus laevis oocytes followed by thin-sectioning electron microscopy (EM) is an excellent system for studying nucleocytoplasmic transport. Because of its large nucleus and high density of nuclear pore complexes (NPCs), nuclear transport can be easily visualized in the Xenopus oocyte. Much insight into the mechanisms of nuclear import and export has been gained through use of this system (reviewed by Pant&eacute;, 2006). In addition, we have used microinjection of Xenopus oocytes to dissect the nuclear import pathways of several viruses that replicate in the host nucleus. 
Here we demonstrate the cytoplasmic microinjection of Xenopus oocytes with a nuclear import substrate. We also show preparation of the injected oocytes for visualization by thin-sectioning EM, including dissection, dehydration, and embedding of the oocytes into an epoxy embedding resin. Finally, we provide representative results for oocytes that have been microinjected with the capsid of the baculovirus Autographa californica nucleopolyhedrovirus (AcMNPV) or the parvovirus Minute Virus of Mice (MVM), and discuss potential applications of the technique.

Here we demonstrate cytoplasmic microinjection of Xenopus laevis oocytes with a nuclear import substrate, as well as preparation of the injected oocytes for visualization by thin-sectioning electron microscopy.

Microinjection of Xenopus laevis oocytes followed by thin-sectioning electron microscopy (EM) is an excellent system for studying nucleocytoplasmic ...

Tissue Determination Using the Animal Cap Transplant (ACT) Assay in Xenopus laevis

Many proteins play a dual role in embryonic development. Those that regulate cell fate determination in a specific tissue can also affect the development of a larger region of the embryo. This makes defining its role in a particular tissue difficult to analyze. For example, noggin overexpression in Xenopus laevis embryos causes the expansion of the entire anterior region, including the eye1,2. From this result, it is not known if Noggin plays a direct role in eye determination or that by causing an expansion of neural tissue, Noggin indirectly affects eye formation. Having this complex phenotype makes studying its eye-specific role in cell fate determination difficult to analyze. We have developed an assay that overcomes this problem. Taking advantage of the pluripotent nature of the Xenopus laevis animal cap 3, we have developed an assay to test the ability of gene product(s), like noggin or the eye field transcription factors (EFTFs), to transform caps into particular tissue or cell types by transplanting this tissue onto the side of the embryo 4. While we have found either Noggin protein treatment or a collection of transcription factors can determine retinal cell fate in animal caps, this procedure could be used to identify gene product(s) involved in specifying other tissues as well.

Tissue Determination Using the Animal Cap Transplant ACT Assay in Xenopus laevis

Animal caps overexpressing gene product(s) are transplanted to the flank of developing Xenopus laevis embryos in order to establish whether tissue is determined.

Many proteins play a dual role in embryonic development. Those that regulate cell fate determination in a specific tissue can also affect the development ...

Live-cell Imaging and Quantitative Analysis of Embryonic Epithelial Cells in Xenopus laevis

Embryonic epithelial cells serve as an ideal model to study morphogenesis where multi-cellular tissues undergo changes in their geometry, such as changes in cell surface area and cell height, and where cells undergo mitosis and migrate. Furthermore, epithelial cells can also regulate morphogenetic movements in adjacent tissues1. A traditional method to study epithelial cells and tissues involve chemical fixation and histological methods to determine cell morphology or localization of particular proteins of interest. These approaches continue to be useful and provide "snapshots" of cell shapes and tissue architecture, however, much remains to be understood about how cells acquire specific shapes, how various proteins move or localize to specific positions, and what paths cells follow toward their final differentiated fate. High resolution live imaging complements traditional methods and also allows more direct investigation into the dynamic cellular processes involved in the formation, maintenance, and morphogenesis of multicellular epithelial sheets. Here we demonstrate experimental methods from the isolation of animal cap tissues from Xenopus laevis embryos to confocal imaging of epithelial cells and simple measurement approaches that together can augment molecular and cellular studies of epithelial morphogenesis.

Live-cell Imaging and Quantitative Analysis of Embryonic Epithelial Cells in Xenopus laevis

Xenopus embryonic epithelia are an ideal model system to study cell behaviors such as polarity development and shape change during epithelial morphogenesis. Traditional histology of fixed samples is increasingly being complemented by live-cell confocal imaging. Here we demonstrate methods to isolate frog tissues and visualize live epithelial cells and their cytoskeleton using live-cell confocal microscopy.

Embryonic epithelial cells serve as an ideal model to study morphogenesis where multi-cellular tissues undergo changes in their geometry, such as changes ...

Production of Xenopus tropicalis Egg Extracts to Identify Microtubule-associated RNAs

Many organisms localize mRNAs to specific subcellular destinations to spatially and temporally control gene expression. Recent studies have demonstrated that the majority of the transcriptome is localized to a nonrandom position in cells and embryos. One approach to identify localized mRNAs is to biochemically purify a cellular structure of interest and to identify all associated transcripts. Using recently developed high-throughput sequencing technologies it is now straightforward to identify all RNAs associated with a subcellular structure. To facilitate transcript identification it is necessary to work with an organism with a fully sequenced genome. One attractive system for the biochemical purification of subcellular structures are egg extracts produced from the frog Xenopus laevis. However, X. laevis currently does not have a fully sequenced genome, which hampers transcript identification. In this article we describe a method to produce egg extracts from a related frog, X. tropicalis, that has a fully sequenced genome. We provide details for microtubule polymerization, purification and transcript isolation. While this article describes a specific method for identification of microtubule-associated transcripts, we believe that it will be easily applied to other subcellular structures and will provide a powerful method for identification of localized RNAs.

Production of Xenopus tropicalis Egg Extracts to Identify Microtubule-associated RNAs

We describe the collection of unfertilized Xenopus tropicalis eggs and production of a meiosis II-arrested egg extract. This egg extract can be used to purify microtubules and microtubule-associated RNAs.

Many  organisms localize mRNAs to specific subcellular destinations to spatially and  temporally control gene expression. Recent studies have demonstrated ...

Facial Transplants in Xenopus laevis Embryos

Craniofacial birth defects occur in 1 out of every 700 live births, but etiology is rarely known due to limited understanding of craniofacial development. To identify where signaling pathways and tissues act during patterning of the developing face, a 'face transplant' technique has been developed in embryos of the frog Xenopus laevis. A region of presumptive facial tissue (the "Extreme Anterior Domain" (EAD)) is removed from a donor embryo at tailbud stage, and transplanted to a host embryo of the same stage, from which the equivalent region has been removed. This can be used to generate a chimeric face where the host or donor tissue has a loss or gain of function in a gene, and/or includes a lineage label. After healing, the outcome of development is monitored, and indicates roles of the signaling pathway within the donor or surrounding host tissues. Xenopus is a valuable model for face development, as the facial region is large and readily accessible for micromanipulation. Many embryos can be assayed, over a short time period since development occurs rapidly. Findings in the frog are relevant to human development, since craniofacial processes appear conserved between Xenopus and mammals.

Facial Transplants in Xenopus laevis Embryos

A technique for transplanting "Extreme Anterior Domain" facial tissue between Xenopus laevis embryos has been developed. Tissue can be moved from one gene expression background into another, allowing the study of local requirements for craniofacial development and for signaling interactions between facial regions.

Craniofacial birth defects occur in 1 out of every 700 live births, but etiology is rarely known due to limited understanding of craniofacial development. ...

Dissection of Xenopus laevis Neural Crest for in vitro Explant Culture or in vivo Transplantation

The neural crest (NC) is a transient dorsal neural tube cell population that undergoes an epithelium-to-mesenchyme transition (EMT) at the end of neurulation, migrates extensively towards various organs, and differentiates into many types of derivatives (neurons, glia, cartilage and bone, pigmented and endocrine cells). In this protocol, we describe how to dissect the premigratory cranial NC from Xenopus laevis embryos, in order to study NC development in vivo and in vitro. The frog model offers many advantages to study early development; abundant batches are available, embryos develop rapidly, in vivo gain and loss of function strategies allow manipulation of gene expression prior to NC dissection in donor and/or host embryos. The NC explants can be plated on fibronectin and used for in vitro studies. They can be cultured for several days in a serum-free defined medium. We also describe how to graft NC explants back into host embryos for studying NC migration and differentiation in vivo.

Dissection of Xenopus laevis Neural Crest for in vitro Explant Culture or in vivo Transplantation

This protocol describes how to dissect premigratory cranial neural crest (NC) from Xenopus laevis neurulas. These explants can be plated on fibronectin and cultured in vitro, or grafted back into host embryos. This technique allows studying the mechanisms of NC epithelium-to-mesenchyme transition, migration, and differentiation.