This work was supported by NIH grants R01 GM075200 and R01 HL083393 (LAT). We acknowledge fellowship support for BAF from NIH T90 DA022871 and the Mallinckrodt Institute of Radiology, and for VDV from grant 09PRE2060795 from the American Heart Association.

1. General Experimental Preparation
<ol>
	<li>Prepare tissue culture medium in the laminar flow hood. 
		<ol>
			<li>Dilute Dulbecco's Modified Eagle's Medium (DMEM)(1 L bottle with 4.5g/L glucose, sodium bicarbonate, and L-glutamine). Add 10 mL penicillin / streptomycin / neomycin antibiotics.</li>
			<li>Remove 100 mL of DMEM with a sterile transfer pipette and replace with 100 mL of chick serum.</li>
			<li>Aliquot the DMEM/10% chick serum/1% antibiotics into sterile 15 mL conical vials and freeze.</li>
		</ol>
	</li>
	<li>Prepare phosphate buffered saline (PBS) supplemented with calcium and magnesium. Mix 100 mL 10X PBS, 900 mL deionized water, and 1 mL of a concentrated calcium and magnesium solution (100 mg/mL CaCl2&bull;2H2O, 100mg/mL MgCl2&bull;6H2O).</li>
	<li>Incubate eggs in a longitudinal orientation to desired stage as defined by Hamburger and Hamilton1 (i.e. 23-25 hours for HH stage 5, or 42-48 hours for HH stages 11-12).</li>
	<li>Remove embryos from the egg using the filter paper carrier method.
		<ol>
			<li>Crack the bottom of the egg on the side of a150 mm petri dish. Pull apart the shell from the bottom and empty the contents of the egg into the dish.</li>
			<li>Remove the thicker, viscous albumen from the egg using blunt forceps.</li>
			<li>Cut circles of Whatman #2 filter paper 2-3 cm. in diameter. Using a single hole punch, punch holes in the middle of the filter paper. Place one of the filter paper rings on the yolk in a manner that keeps the embryo visible through the central hole in the ring.</li>
			<li>Cut around the outer diameter of the filter paper with microscissors. Remove the paper from the yolk with fine forceps.</li>
			<li>Gently rinse the embryo in PBS to remove adherent yolk particles. (HH5 embryos may need to soak as long as 15-20 minutes to remove residual yolk). Remove the filter paper assembly from the bath and place it ventral side up in a 35 mm culture dish. Place a second filter paper ring atop the first, sandwiching the embryo between the rings. Put a metal ring around the outside of the filter paper sandwich (&asymp; 1.5 - 2 cm diameter) to secure the embryo.</li>
			<li>Submerge the embryo in culture medium. Verify proper morphology and embryonic stage with a bright field microscope.</li>
		</ol>
	</li>
	<li>Use a pipette puller to prepare fine tip glass needles (1.5 mm inner diameter) (World Precision Instruments, Sarasota FL) for subsequent tissue dissections and labeling. </li>
</ol>
2. DiI Labeling of HH Stage 5 Embryos using Magnetic Iron Particles
<ol>
<li>Add a few drops of saturated DiI in alcohol to a small quantity of iron powder (iron reduced, Mallinckrodt Baker, Inc., Phillipsburg, NJ) at room temperature and let dry. (This usually causes the particles to cake together once the alcohol has evaporated.) Use the tip of a pulled micropipette to break up the clumped iron particles, transfer them to a microcentrifuge tube, and rinse them several times and cover with deionized water.</li>
<li>To label ectodermal cells in a HH stage 5 embryo, place the harvested embryo (i.e., the entire filter paper assembly) dorsal side up in a 35 mm culture dish covered with a generous layer of culture media and put the dish under a dissecting microscope. Use a glass needle to remove the vitelline membrane and expose the ectodermal cells. (Take care to not pierce embryo.)</li>
<li>Draw a column of labeled iron particles (in deionized water) into a Pasteur pipette. Using the dissecting microscope, submerge the pipette tip beneath the fluid surface and position just above the embryo. Rolling the pipette slowly back and forth between the fingertips, jostle the particles out of the pipiette and sprinkle them across the embryo.</li>
<li>Once the embryo is covered with particles, carefully place it in a 37&deg;C incubator for approximately 10 minutes. Take embryo out of the incubator and use a strong magnet to remove the particles.</li>
<li>Use a fluorescent microscope with attached video camera and appropriate filter settings to acquire both bright field and fluorescent images of the embryo, as shown in Figure 2A,B.</li>
<li>Note the label density. If a denser distribution is desired, use additional particles (as described above) to label the embryo again and extend the incubation time accordingly. If the labels are too dense, add a small quantity of unlabeled iron particles to the microcentrifuge tube filled with already labeled particles and mix the tube contents. Repeat the labeling process with a new embryo and again note label density.</li>
</ol>
3. Polystyrene Microsphere Labeling for Optical Coherence Tomography (OCT) Imaging of HH11-12 embryos
<ol>
<li>Prepare a solution of black 10 &mu;m diameter microspheres (Polysciences Inc., Warrington, PA) in PBS in a 35 mm petri dish. (Beads should be large enough to generate contrast for OCT imaging, but small enough such that bead size is on the order of individual cells. Specific values will vary depending on the resolution of your OCT system and the tissues being studied). One drop of the stock solution (2.6% solids-latex) per mL of PBS is generally sufficient. Pull this solution into a 10cc syringe using a 22 gauge needle. Vortex the solution for 30 seconds to ensure a uniform bead distribution.</li>
<li>Bevel the tips of your pulled glass needles using a computer hard drive2. Tip diameter should be large enough that beads can fit through the opening, but at the same time, as small as possible to minimize tissue wounding during injection.</li>
<li>Fill a beveled glass needle with the bead solution using the syringe. Lightly flick the micropipette to ensure that the solution has reached the tip. </li>
<li>Using a micromanipulator, place the glass needle in the same field of view as the embryo. Beads should be flowing out of the pipette tip.<ol>
<li>If no beads exit the micropipette, there may be a clog. In this case, repeat steps 3.2-3.4 with a new glass needle.</li>
<li>If too many beads cloud your field of view, your pipette tip may be too wide. Repeat steps 3.2-3.4 with a new pipette and a new embryo.</li></ol></li>
<li>Insert the pipette tip into the lumen of the brain tube. Be sure not to pierce the ventral floor of the tissue. You will see a transient swelling of the tissue as beads and fluid enter the lumen. This indicates a successful intubation; now, quickly remove the glass needle.</li>
<li>Verify a sufficient bead density in the inner lumen of the brain tube using a bright field microscope. Let the beads settle for 20 - 30 minutes before proceeding to Sect. 4.</li>
</ol>
4. Data Acquisition
* The following methods are well suited for the fluorescence labeling technique described above. However, with the microsphere labeling technique, beads can dislodge when transferring samples between incubators and imaging systems. In this case it is best to proceed to step 4.2 (which avoids sample movement/agitation altogether). In both methods, embryos are cultured while submerged in liquid culture medium. If the embryo is not completely submerged (as is the case with some conventional tissue culture techniques), tissue geometry is greatly altered by abnormally high surface tension loads and strain distributions will fail to accurately capture normal 3-D morphogenesis3, 4. Moreover, submerging the tissue prevents the bright contrast observed at the liquid-gas interface during OCT imaging from obscuring embryonic morphology and marker coordinates. 
<ol>
<li>Embryo Culture (general)<ol>
<li>After acquiring your first images (bright field, fluorescence, or OCT), put up to seven 35 mm culture dishes (with embryos) in a 150 mm Petri dish and place the entire assembly in a small plastic bag. Add a couple of drops of deionized water to the bag for humidification and fill the bag with a 95% O2 / 5% CO2 mixture. Place the embryos into the incubator at 37&deg;C (Queue Stabiletherm, Asheville, NC) for the desired time until the next imaging timepoint. Using this method, experiments can be performed on multiple embryos in the same day.</li></ol></li>
<li>Time-lapse culture (automated)<ol>
<li>Place your labeled embryos into a Delta T Dish (35 mm outer diameter, 23 mm central aperture with tapered side wall, 0.17mm thick glass; Bioptechs, Butler, PA) containing 1.2 mL of culture medium.</li>
<li>Cover the dish with a glass lid kept at 37&deg;C using a Bioptechs Delta T4 Culture Dish Controller (to prevent condensation). Superfuse the embryo with a 95% O2 / 5% CO2 gas mixture supplied from a Mini-Pump Variable Flow device (Fisher Scientific). Prior to reaching the embryo, heat and humidify the gas by bubbling it through deionized water on a warm (&lt;100&deg;C) hot plate. A schematic of this experimental set-up is shown in Figure 1.</li>
<li>Set up the software to automatically acquire images at user-specified time intervals. For our fluorescence microscope, we use Openlab software (PerkinElmer, Waltham, MA), and for our OCT system, we use swept source Thorlabs software (Newton, NJ).Because this method is currently only capable of handling one time-lapse experiment at a time, it is often best to allow these types of cultures to run overnight. One could circumvent this limitation by installing an automated translatable stage compatible with both the hardware and software of the imaging system5.</li></ol></li>
</ol>
5. Representative Results:
Labels are tracked automatically (using Volocity, PerkinElmer) or manually (using the Manual Tracking plugin in ImageJ, NIH) in each experiment. In the fluorescent labeling technique, we use the Matlab routine gridfit to fit 2D surfaces through the marker coordinates, which enables morphogenetic surface strains to be calculated6, 7. Standard equations are used to transform these values into a relevant embryonic coordinate system. Alternatively, in the OCT technique, surfaces are generated from segmented image volumes (obtained via standard software such as Matlab or Caret8) and strains can be calculated in the direction of maximum or minimum curvature of the sample9. 
We used our iron particle technique to label and track the motion of ectodermal cells during head fold formation in the early chick embryo. As depicted in Figure 2A, fluorescently labeled cells were distributed across the entire embryo. Bright field and fluorescent images of the embryo were captured at different intervals during ex ovo culture. The motions of the tracked labels (Fig. 2B,C) were then used to calculate the evolving morphogenetic strain distributions during head fold formation (Fig. 2D-F).
Similarly, our polystyrene microsphere technique was used to track tissue movements at the mid-hindbrain boundary of the early chick brain. As shown in Figure 3 B-D, bead motions were tracked for 6 hours and strains characterizing tissue deformation in the longitudinal and circumferential directions were calculated from marker coordinates. Note that this method is capable of handling distinctly 3-D deformations as beads tend to stick to all sides of the inner lumen of the brain (Fig. 3A). 
<img src="/files/ftp_upload/3129/3129fig1.jpg" alt="Figure 1"/> Figure 1. Schematic of set-up for time-lapse tissue culture.
<img src="/files/ftp_upload/3129/3129fig2.jpg" alt="Figure 2"/> Figure 2. Quantifying 2-D tissue deformations in the early chick embryo using tracked fluorescent labels. (A) Merged bright field/fluorescent image of HH stage 5 embryo after removal of iron particles. DiI-labeled ectodermal cells (red) are distributed across the entire embryo. (B,C) The motion of labeled cells was tracked using ImageJ. Label displacements were calculated in embryo coordinates (X,Y). Note that A and B are the same image. (D-F) Contour plots of evolving longitudinal strain distributions during head fold formation. Longitudinal Lagrangian strains were calculated relative to HH stage 5 (i.e., A and B) after (D) 75 min, (E) 120 min, and (F) 165 min of incubation. These strains characterize the length changes of line elements originally oriented along the Y-axis (in the stage 5 embryo). Further details on using tracked label coordinates to calculate morphogenetic strains can be found in Filas et al. (2007)6 and Varner et al. (2010)7. Note that C and F are the same image. Scale bar = 500 &mu;m.
<img src="/files/ftp_upload/3129/3129fig3.jpg" alt="Figure 3"/> Figure 3. Quantifying 3-D tissue deformations in the early chick brain. (A) Polystyrene microspheres that adhere to the dorsal (black arrow) and ventral (white arrow) sides of the brain tube are easily discernible from surrounding tissues in transverse cross section.Ventral views of OCT reconstructions show microsphere locations near the mid-hindbrain boundary at (B) HH12 and after (C) 6 hr of incubation (M: midbrain, H: hindbrain). Several groups of beads are outlined to highlight the overall deformation of the tissue. (D) Longitudinal (Ezz) and circumferential (E&theta;&theta;) Lagrangian strains were calculated from these deformations. Positive longitudinal and negative circumferential strains at the mid-hindbrain boundary reflect an axial lengthening and circumferential shortening of this region during culture. Further details on calculating morphogenetic strains for complex surfaces during morphogenesis can be found in Filas et al. (2008)9. Scale bar = 200 &mu;m.

<ol>
	<li>Hamburger, V., Hamilton, H. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+series+of+normal+stages+in+the+development+of+the+chick+embryo.">A series of normal stages in the development of the chick embryo.</a> Journal of Morphology. 88, 49-92 (1951).</li><li>Canfield, J. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dry+beveling+micropipettes+using+a+computer+hard+drive.">Dry beveling micropipettes using a computer hard drive.</a> J. Neurosci. Methods. 158, 19-21 (2006).</li><li>Voronov, D. A., Taber, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cardiac+looping+in+experimental+conditions:+the+effects+of+extraembryonic+forces.">Cardiac looping in experimental conditions: the effects of extraembryonic forces.</a> Developmental Dynamics. 224, 413-421 (2002).</li><li>Filas, B. A., Bayly, P. V., Taber, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanical+stress+as+a+regulator+of+cytoskeletal+contractility+and+nuclear+shape+in+embryonic+epithelia.">Mechanical stress as a regulator of cytoskeletal contractility and nuclear shape in embryonic epithelia.</a> Ann. Biomed. Eng. 39, 443-454 (2011).</li><li>Kozel, B. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Elastic+fiber+formation:+a+dynamic+view+of+extracellular+matrix+assembly+using+timer+reporters.">Elastic fiber formation: a dynamic view of extracellular matrix assembly using timer reporters.</a> J. Cell. Physiol. 207, 87-96 (2006).</li><li>Filas, B. A., Efimov, I. R., Taber, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optical+coherence+tomography+as+a+tool+for+measuring+morphogenetic+deformation+of+the+looping+heart.">Optical coherence tomography as a tool for measuring morphogenetic deformation of the looping heart.</a> Anat. Rec. 290, 1057-1068 (2007).</li><li>Varner, V. D., Voronov, D. A., Taber, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanics+of+head+fold+formation:+investigating+tissue-level+forces+during+early+development.">Mechanics of head fold formation: investigating tissue-level forces during early development.</a> Development. 137, 3801-3811 (2010).</li><li>Van Essen, D. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+integrated+software+suite+for+surface-based+analyses+of+cerebral+cortex.">An integrated software suite for surface-based analyses of cerebral cortex.</a> J. Am. Med. Inform. Assoc. 8, 443-459 (2001).</li><li>Filas, B. A., Knutsen, A. K., Bayly, P. V., Taber, L. 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+new+method+for+measuring+deformation+of+folding+surfaces+during+morphogenesis.">A new method for measuring deformation of folding surfaces during morphogenesis.</a> J. Biomech. Eng. 130, 061010-061010 (2008).</li><li>Yuan, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Co-registered+optical+coherence+tomography+and+fluorescence+molecular+imaging+for+simultaneous+morphological+and+molecular+imaging.">Co-registered optical coherence tomography and fluorescence molecular imaging for simultaneous morphological and molecular imaging.</a> Phys. Med. Biol. 55, 191-206 (2010).</li><li>Zamir, E. A., Czirok, A., Rongish, B. J., Little, C. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+digital+image-based+method+for+computational+tissue+fate+mapping+during+early+avian+morphogenesis.">A digital image-based method for computational tissue fate mapping during early avian morphogenesis.</a> Ann. Biomed. Eng. 33, 854-865 (2005).</li><li>Blanchard, G. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tissue+tectonics:+morphogenetic+strain+rates,+cell+shape+change+and+intercalation.">Tissue tectonics: morphogenetic strain rates, cell shape change and intercalation.</a> Nat. Methods. 6, 458-464 (2009).</li></ol>

No conflicts of interest declared.

Two tissue labeling techniques are presented for the ex ovo culture of early chick embryos. The first uses fluorescent lipophilic dyes delivered via magnetic iron particles to simultaneously label hundreds of cells. However, this method is currently not compatible with optical coherence tomography, as fluorescent dyes generally show little contrast from surrounding tissues using OCT10. Hence, we show an alternative technique using polystyrene microspheres to label tissues for time-lapse OCT analysis. ...

<table border="1">
 <table border="1">
 <tbody>
 <tr>
 <td>Material / Reagent </td>
 <td>Company</td>
 <td>Catalogue number</td>
 <td>Comments </td>
 </tr>
 
 <tr>
 <td>DMEM &#x2013; high glucose</td>
 <td>Sigma-Aldrich</td>
 <td>D5796</td>
 <td>&nbsp;</td>
 </tr>
 
 <tr>
 <td>Penicillin/Streptomycin/Neomycin</td>
 <td>Sigma-Aldrich</td>
 <td>P4083</td>
 <td>&nbsp;</td>
 </tr>
 
 <tr>
 <td>Chicken Serum</td>
 <td>Invitrogen</td>
 <td>16110-082</td>
 <td>&nbsp;</td>
 </tr>
 
 <tr>
 <td>Dulbecco&#x2019;s Phosphate Buffered Saline</td>
 <td>Sigma-Aldrich</td>
 <td>D1408</td>
 <td>10X</td>
 </tr>
 
 <tr>
 <td>Whatman #2 Filter Paper</td>
 <td>Whatman</td>
 <td>1002 090</td>
 <td>90mm diameter</td>
 </tr>
 
 <tr>
 <td>Glass Micropipettes</td>
 <td>World Precision Instruments (WPI)</td>
 <td>TW150-6</td>
 <td>1.5mm inner diameter</td>
 </tr>
 
 <tr>
 <td>DiI</td>
 <td>Invitrogen</td>
 <td>D-282</td>
 <td>&nbsp;</td>
 </tr>
 
 <tr>
 <td>Iron Reduced</td>
 <td>Mallinckrodt Baker Inc.</td>
 <td>5320</td>
 <td>&nbsp;</td>
 </tr>
 
 <tr>
 <td>10 &mu;m Diameter Microspheres (black)</td>
 <td>Polysciences Inc.</td>
 <td>24294</td>
 <td>&nbsp;</td>
 </tr>
 
 <tr>
 <td>Delta T Dish (for time lapse culture)</td>
 <td>Bioptechs</td>
 <td>04200415B</td>
 <td>0.17mm thick, black</td>
 </tr>
 
 <tr>
 <td>Delta T4 Culture Dish Controller</td>
 <td>Bioptechs</td>
 <td>0420-4-03</td>
 <td>&nbsp;</td>
 </tr>
 
 <tr>
 <td>Mini-Pump Variable Flow Device</td>
 <td>Fisher Scientific</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 </tbody>
 </table>

tracking morphogenetic tissue deformations in the early chick embryo

Embryonic epithelia undergo complex deformations (e.g. bending, twisting, folding, and stretching) to form the primitive organs of the early embryo. Tracking fiducial markers on the surfaces of these cellular sheets is a well-established method for estimating morphogenetic quantities such as growth, contraction, and shear. However, not all surface labeling techniques are readily adaptable to conventional imaging modalities and possess different advantages and limitations. Here, we describe two labeling methods and illustrate the utility of each technique. In the first method, hundreds of fluorescent labels are applied simultaneously to the embryo using magnetic iron particles. These labels are then used to quantity 2-D tissue deformations during morphogenesis. In the second method, polystyrene microspheres are used as contrast agents in non-invasive optical coherence tomography (OCT) imaging to track 3-D tissue deformations. These techniques have been successfully implemented in our lab to studythe physical mechanisms of early head fold, heart, and brain development, and should be adaptable to a wide range morphogenetic processes.

Watch this Scientific Journal Video about Tracking Morphogenetic Tissue Deformations in the Early Chick Embryo at JoVE.com

Tracking Morphogenetic Tissue Deformations in the Early Chick Embryo

This article describes surface labeling and ex ovo tissue culture in the early chick embryo. Techniques amenable to time-lapse bright field, fluorescence, and optical coherence tomography imaging are presented. Tracking surface labels with high spatiotemporal resolution enables kinematic quantities such as morphogenetic strains (deformations) to be calculated in both two and three dimensions.

Embryonic epithelia undergo complex deformations (e.g. bending, twisting, folding, and stretching) to form the primitive organs of the early embryo. ...

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12.9K Views.  Washington University. This article describes surface labeling and ex ovo tissue culture in the early chick embryo. Techniques amenable to time-lapse bright field, fluorescence, and optical coherence tomography imaging are presented. Tracking surface labels with high spatiotemporal resolution enables kinematic quantities such as morphogenetic strains (deformations) to be calculated in both two and three dimensions.

Video: Tracking Morphogenetic Tissue Deformations in the Early Chick Embryo

Method for Culture of Early Chick Embryos ex vivo (New Culture)

The chick embryo is a valuable tool in the study of early embryonic development. Its transparency, accessibility and ease of manipulation, make it an ideal tool for studying  the formation and patterning of brain, neural tube, somite and heart primordia. Applications of chick embryo culture include electroporation of DNA or RNA constructs in order to analyze gene function, grafts of growth factor coated beads such as FGFs and BMPs , as well as whole mount in situ hybridization and immunohistochemistry. This video demonstrates the different steps in chick embryo culture; First, the embryo is explanted in saline. Then, the embryo is centered on a glass ring. The membranes surrounding the embryo are lifted along the walls of the ring. The ring is then placed in a culture dish containing a pool of albumine. The culture dish is sealed and placed in a humid chamber, where the embryo is cultured for up to 24 hrs. Finally, the embryo is removed from the ring, fixed and processed for further applications. A troubleshooting guide is also presented.

Method for Culture of Early Chick Embryos ex vivo New Culture

This video demonstrates New culture, a method by which chick embryos are cultured outside the egg for up to 24 hr. This method enables one to study early development (primitive streak to 14 som.), a period corresponding to E7-9 in mouse. Applications of this technique include electroporation, in situ hybridization and immunohistochemistry.

The chick embryo is a valuable tool in the study of early embryonic development. Its transparency, accessibility and ease of manipulation, make it an ...

Double Whole Mount in situ Hybridization of Early Chick Embryos

The chick embryo is a valuable tool in the study of early embryonic development. Its transparency, accessibility and ease of manipulation, make it an ideal tool for studying  gene expression in brain, neural tube, somite and heart primordia formation. This video demonstrates the different steps in 2-color whole mount in situ hybridization; First, the embryo is dissected from the egg and fixed in paraformaldehyde. Second, the embryo is processed for prehybridization. The embryo is then hybridized with two different probes, one coupled to DIG, and one coupled to FITC.  Following overnight hybridization, the embryo is incubated with DIG coupled antibody. Color reaction for DIG substrate is performed, and the region of interest appears blue. The embryo is then incubated with FITC coupled antibody. The embryo is processed for color reaction with FITC, and the region of interest appears red. Finally, the embryo is fixed and processed for phtograph and sectioning. A troubleshooting guide is also presented.

This video demonstrates 2-color whole mount in situ hybridization, a method by which the spatial and temporal expression pattern of 2 different genes can be visualized in young chick embryos. This method was originally introduced by David Wilkinson, Domingos Henrique, Phil Ingham and David Ish -Horowicz.

Assay for Neural Induction in the Chick Embryo

The chick embryo is a valuable tool in the study of early embryonic development. Its transparency, accessibility and ease of manipulation, make it an ideal tool for studying the formation and initial patterning of the nervous system. This video demonstrates how to graft organizer tissue into a host, a method by which Hensen s node (the organizer in the chick embryo) is grafted to a host competent ectoderm. The organizer graft instructs overlying na ve tissue to adopt a neural fate via neural inducing signals. This mechanism is referred to as neural induction, and constitutes the initial step in the formation of brain and spinal cord in amniotes. This method is essentially used for the characterization of putative neural inducing molecules in chick. This video demonstrates the different steps in the assay for neural induction; First, the donnor embryo is explanted and pinned on a dish. Then, the host embryo is prepared for New culture. The graft is excised and transplanted to the host area pellucida margin. The host is cultured for 18-22 hrs. The assembly is fixed and processed for further applications (e.g. in situ hybridization). This method was originally devised by Waddington 1,2 and Gallera 3,4.

Neural induction is the first step in the formation of the brain. It is a mechanism by which Hensen's node (organizer), instructs adjacent tissue to adopt a neural fate, i.e. to give rise to the nervous system. This video demonstrates an assay for neural induction in chick embryo.

Electrophysiological Recording in the Drosophila Embryo

Drosophila is a premier genetic model for the study of both embryonic development and functional neuroscience. Traditionally, these fields are quite isolated from each other, with largely independent histories and scientific communities. However, the interface between these usually disparate fields is the developmental programs underlying acquisition of functional electrical signaling properties and differentiation of functional chemical synapses during the final phases of neural circuit formation. This interface is a critically important area for investigation. In Drosophila, these phases of functional development occur during a period of &lt;8 hours (at 25&deg;C) during the last third of embryogenesis. This late developmental period was long considered intractable to investigation owing to the deposition of a tough, impermeable epidermal cuticle. A breakthrough advance was the application of water-polymerizing surgical glue that can be locally applied to the cuticle to enable controlled dissection of late-stage embryos. With a dorsal longitudinal incision, the embryo can be laid flat, exposing the ventral nerve cord and body wall musculature to experimental investigation. Whole-cell patch-clamp techniques can then be employed to record from individually-identifiable neurons and somatic muscles. These recording configurations have been used to track the appearance and maturation of ionic currents and action potential propagation in both neurons and muscles. Genetic mutants affecting these electrical properties have been characterized to reveal the molecular composition of ion channels and associated signaling complexes, and to begin exploration of the molecular mechanisms of functional differentiation. A particular focus has been the assembly of synaptic connections, both in the central nervous system and periphery. The glutamatergic neuromuscular junction (NMJ) is most accessible to a combination of optical imaging and electrophysiological recording. A glass suction electrode is used to stimulate the peripheral nerve, with excitatory junction current (EJC) recordings made in the voltage-clamped muscle. This recording configuration has been used to chart the functional differentiation of the synapse, and track the appearance and maturation of presynaptic glutamate release properties. In addition, postsynaptic properties can be assayed independently via iontophoretic or pressure application of glutamate directly to the muscle surface, to measure the appearance and maturation of the glutamate receptor fields. Thus, both pre- and postsynaptic elements can be monitored separately or in combination during embryonic synaptogenesis. This system has been heavily used to isolate and characterize genetic mutants that impair embryonic synapse formation, and thus reveal the molecular mechanisms governing the specification and differentiation of synapse connections and functional synaptic signaling properties.

Electrophysiological Recording in the Drosophila Embryo

Electrophysiological recordings from Drosophila embryos allow analyses of developing muscle and neuron electrical properties, as well as characterization of functional synaptogenesis at the glutamatergic neuromuscular junction and central cholinergic and GABAergic synapses.

Drosophila is a premier genetic model for the study of both embryonic development and functional neuroscience. Traditionally, these fields are quite ...

Quantitation of &gamma;H2AX Foci in Tissue Samples

DNA double-strand breaks (DSBs) are particularly lethal and genotoxic lesions, that can arise either by endogenous (physiological or pathological) processes or by exogenous factors, particularly ionizing radiation and radiomimetic compounds. Phosphorylation of the H2A histone variant, H2AX, at the serine-139 residue, in the highly conserved C-terminal SQEY motif, forming &gamma;H2AX, is an early response to DNA double-strand breaks1. This phosphorylation event is mediated by the phosphatidyl-inosito 3-kinase (PI3K) family of proteins, ataxia telangiectasia mutated (ATM), DNA-protein kinase catalytic subunit and ATM and RAD3-related (ATR)2. Overall, DSB induction results in the formation of discrete nuclear &gamma;H2AX foci which can be easily detected and quantitated by immunofluorescence microscopy2. Given the unique specificity and sensitivity of this marker, analysis of &gamma;H2AX foci has led to a wide range of applications in biomedical research, particularly in radiation biology and nuclear medicine. The quantitation of &gamma;H2AX foci has been most widely investigated in cell culture systems in the context of ionizing radiation-induced DSBs. Apart from cellular radiosensitivity, immunofluorescence based assays have also been used to evaluate the efficacy of radiation-modifying compounds. In addition, &gamma;H2AX has been used as a molecular marker to examine the efficacy of various DSB-inducing compounds and is recently being heralded as important marker of ageing and disease, particularly cancer3. Further, immunofluorescence-based methods have been adapted to suit detection and quantitation of &gamma;H2AX foci ex vivo and in vivo4,5. Here, we demonstrate a typical immunofluorescence method for detection and quantitation of &gamma;H2AX foci in mouse tissues.

Quantitation of &#947;H2AX Foci in Tissue Samples

Quantitation of DNA double-strand breaks on the basis of &gamma;H2AX foci has become an invaluable tool, particularly in radiation biology, for the evaluation of tissue radiosensitivity and effects of radiation modifying compounds. Here we demonstrate the use of an immunofluorescence assay for quantitation of &gamma;H2AX foci in tissue samples.

DNA double-strand breaks (DSBs) are particularly lethal and genotoxic lesions, that can arise either by endogenous (physiological or pathological) ...

Single Cell Transfection in Chick Embryos

A central theme in developmental biology is the diversification of lineages and the elucidation of underlying molecular mechanisms. This entails a thorough analysis of the fates of single cells under normal and experimental conditions. To this end, transfection methods that target single progenitors are a prerequisite. We describe here a technically straightforward method for transfecting single cells in chicken tissues in-ovo, allowing reliable lineage tracing as well as genetic manipulation. Specific tissue domains are targeted within the somite or neural tube, and DNA is injected directly into the epithelium of interest, resulting in sporadic transfection of single cells. Using reporters, clonal populations may consequently be traced for up to three days, and behavior of genetically manipulated clonal populations can be compared with that of controls. This method takes advantage of the accessibility of the chick embryo along with emerging tools for genetic manipulation. We compare and discuss its advantages over the widely-used electroporation method, and possible applications and use in additional in-vivo models are also suggested. We advocate the use of this method as a significant addition and complement for existing lineage tracing and genetic interference tools.

Using fine tip micropipettes we inject plasmid DNA into subdomains of chicken somites or neural tubes. The concentration of the plasmid is adjusted to generate single transfected cells. We then allow the cells to develop into clonal populations.

A central theme in developmental biology is the diversification of lineages and the elucidation of underlying molecular mechanisms. This entails a ...

Chromatin Immunoprecipitation Assay for Tissue-specific Genes using Early-stage Mouse Embryos

Chromatin immunoprecipitation (ChIP) is a powerful tool to identify protein:chromatin interactions that occur in the context of living cells 1-3. This technique has been widely exploited in tissue culture cells, and to a lesser extent, in primary tissue. The application of ChIP to rodent embryonic tissue, especially at early times of development, is complicated by the limited amount of tissue and the heterogeneity of cell and tissue types in the embryo. Here we present a method to perform ChIP using a dissociated embryonic day 8.5 (E8.5) embryo. Sheared chromatin from a single E8.5 embryo can be divided into up to five aliquots, which allows the investigator sufficient material for controls and for investigation of specific protein:chromatin interactions.
We have utilized this technique to begin to document protein:chromatin interactions during the specification of tissue-specific gene expression programs. The heterogeneity of cell types in an embryo necessarily restricts the application of this technique because the result is the detection of protein:chromatin interactions without distinguishing whether the interactions occur in all, a subset of, or a single cell type(s). However, examination of tissue-specific genes during or following the onset of tissue-specific gene expression is feasible for two reasons. First, immunoprecipitation of tissue specific factors necessarily isolates chromatin from the cell type where the factor is expressed. Second, immunoprecipitation of coactivators and histones containing post-translational modifications that are associated with gene activation should only be found at genes and gene regulatory sequences in the cell type where the gene is being or has been activated. The technique should be applicable to the study of most tissue-specific gene activation events.
In the example described below, we utilized E8.5 and E9.5 mouse embryos to examine factor binding at a skeletal muscle specific gene promoter. Somites, which are the precursor tissues from which the skeletal muscles of the trunk and limbs will form, are present at E8.5-9.54,5. Myogenin is a regulatory factor required for skeletal muscle differentiation 6-9. The data demonstrate that myogenin is associated with its own promoter in E8.5 and E9.5 embryos. Because myogenin is only expressed in somites at this stage of development 6,10, the data indicate that myogenin interactions with its own promoter have already occurred in skeletal muscle precursor cells in E8.5 embryos.

We demonstrate a chromatin immunoprecipitation (ChIP) method to identify factor interactions at tissue-specific genes during or after the onset of tissue-specific gene expression in mouse embryonic tissue. This protocol should be widely applicable for the study of tissue-specific gene activation as it occurs during normal embryonic development.

Chromatin immunoprecipitation (ChIP) is a powerful tool to identify protein:chromatin interactions that occur in the context of living cells 1-3. This ...

Time-lapse Microscopy of Early Embryogenesis in Caenorhabditis elegans

Caenorhabditis elegans has often been used as a model system in studies of early developmental processes. The transparency of the 
embryos, the genetic resources, and the relative ease of transformation are qualities that make C. elegans an excellent model for early embryogenesis. Laser-based 
confocal microscopy and fluorescently labeled tags allow researchers to follow specific cellular structures and proteins in the developing embryo. For example, 
one can follow specific organelles, such as lysosomes or mitochondria, using fluorescently labeled dyes. These dyes can be delivered to the early embryo by means 
of microinjection into the adult gonad. Also, the localization of specific proteins can be followed using fluorescent protein tags. Examples are presented here 
demonstrating the use of a fluorescent lysosomal dye as well as fluorescently tagged histone and ubiquitin proteins. The labeled histone is used to visualize 
the DNA and thus identify the stage of the cell cycle. GFP-tagged ubiquitin reveals the dynamics of ubiquitinated vesicles in the early embryo. Observations 
of labeled lysosomes and GFP:: ubiquitin can be used to determine if there is colocalization between ubiquitinated vesicles and lysosomes. A technique for 
the microinjection of the lysosomal dye is presented. Techniques for generating transgenenic strains are presented elsewhere (1, 2). For imaging, embryos 
are cut out of adult hermaphrodite nematodes and mounted onto 2% agarose pads followed by time-lapse microscopy on a standard laser scanning confocal 
microscope or a spinning disk confocal microscope. This methodology provides for the high resolution visualization of early embryogenesis.

Time-lapse Microscopy of Early Embryogenesis in Caenorhabditis elegans

This article describes a technique for the visualization of the early events of embryogenesis in the nematode Caenorhabditis elegans.

Caenorhabditis elegans has often been used as a model system in studies of early developmental processes.  The transparency of the 
embryos, the genetic ...

In Ovo Electroporation in Embryonic Chick Retina

Chicken embryonic retina is an excellent tool to study retinal development in higher vertebrates. Because of large size and external development, it is comparatively very easy to manipulate the chick embryonic retina using recombinant DNA/RNA technology. Electroporation of DNA/RNA constructs into the embryonic retina have a great advantage to study gene regulation in retinal stem/progenitor cells during retinal development. Different type of assays such as reporter gene assay, gene over-expression, gene knock down (shRNA) etc. can be performed using the electroporation technique. This video demonstrates targeted retinal injection and in ovo electroporation into the embryonic chick retina at the Hamburger and Hamilton stage 22-23, which is about embryonic day 4 (E4). Here we show a rapid and convenient in ovo electroporation technique whereby a plasmid DNA that expresses green fluorescent protein (GFP) as a marker is directly delivered into the chick embryonic subretinal space and followed by electric pulses to facilitate DNA uptake by retinal stem/progenitor cells. The new method of retinal injection and electroporation at E4 allows the visualization of all retinal cell types, including the late-born neurons1, which has been difficult with the conventional method of injection and electroporation at E1.52.

In Ovo Electroporation in Embryonic Chick Retina

The overall goal of this video is to show how to perform targeted retinal injection and in ovo electroporation of DNA/RNA constructs into the chick embryonic retina at the Hamburger and Hamilton stage 22-23, which is about embryonic day 4 (E4). This technique is very useful to study gene expression, gene regulation, and morphological change in developing chick retina.

Chicken embryonic retina is an excellent tool to study retinal development in higher vertebrates. Because of large size and external development, it is ...

Mouse Embryonic Development in a Serum-free Whole Embryo Culture System

Mid-gestation stage mouse embryos were cultured utilizing a serum-free culture medium prepared from commercially available stem cell media supplements in an oxygenated rolling bottle culture system. Mouse embryos at E10.5 were carefully isolated from the uterus with intact yolk sac and in a process involving precise surgical maneuver the embryos were gently exteriorized from the yolk sac while maintaining the vascular continuity of the embryo with the yolk sac. Compared to embryos prepared with intact yolk sac or with the yolk sac removed, these embryos exhibited superior survival rate and developmental progression when cultured under similar conditions. We show that these mouse embryos, when cultured in a defined medium in an atmosphere of 95% O2 / 5% CO2 in a rolling bottle culture apparatus at 37 &#176;&#8203;C for 16-40 hr, exhibit morphological growth and development comparable to the embryos developing in utero. We believe this method will be useful for investigators needing to utilize whole embryo culture to study signaling interactions important in embryonic organogenesis.

Serum utilized in embryo cultures contains unknown components that can affect the outcome of experiments especially in studies involving signaling interactions. Here we utilized a serum-free oxygenated culture system and show that mid-gestation mouse embryos cultured for 16-40 hr&#160;exhibit morphological development comparable to embryos developing in utero.

Mid-gestation stage mouse embryos were cultured utilizing a serum-free culture medium prepared from commercially available stem cell media supplements in ...