Name: tracking morphogenetic tissue deformations in the early chick embryo
Uploaded: 2011-10-17T12:00:00
Description: Watch this Scientific Journal Video about Tracking Morphogenetic Tissue Deformations in the Early Chick Embryo at JoVE.com

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>
	</l...

<ol>
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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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