Tracking Morphogenetic Tissue Deformations in the Early Chick Embryo

Summary

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.

Abstract

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.

Protocol

1. General Experimental Preparation

1. Prepare tissue culture medium in the laminar flow hood.
   1. 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.
   2. Remove 100 mL of DMEM with a sterile transfer pipette and replace with 100 mL of chick serum.
   3. Aliquot the DMEM/10% chick serum/1% antibiotics into sterile 15 mL conical vials and freeze.
2. Prepare phosphate buffered saline (PBS) supplemented with calcium and magnesium. Mix 100 mL 10X PBS, 900 mL deionized water, and 1 mL of a con....

Discussion

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. .......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
DMEM — high glucose	Sigma-Aldrich	D5796
Penicillin/Streptomycin/Neomycin	Sigma-Aldrich	P4083
Chicken Serum	Invitrogen	16110-082
Dulbecco’s Phosphate Buffered Saline	Sigma-Aldrich	D1408	10X
Whatman #2 Filter Paper	Whatman, GE Healthcare	1002 090	90mm diameter
Glass Micropipettes	World Precision Instruments, Inc.	TW150-6	1.5mm inner diameter
DiI	Invitrogen	D-282
Iron Reduced	Mallinckrodt Baker Inc.	5320
10 μm Diameter Microspheres (black)	Polysciences, Inc.	24294
Delta T Dish (for time lapse culture)	Bioptechs	04200415B	0.17mm thick, black
Delta T4 Culture Dish Controller	Bioptechs	0420-4-03
Mini-Pump Variable Flow Device	Fisher Scientific