Transplantation of GFP-expressing Blastomeres for Live Imaging of Retinal and Brain Development in Chimeric Zebrafish Embryos

Summary

We demonstrate a protocol to generate chimeric zebrafish embryos for live imaging cellular behavior during embryogenesis.

Abstract

Cells change extensively in their locations and property during embryogenesis. These changes are regulated by the interactions between the cells and their environment. Chimeric embryos, which are composed of cells of different genetic background, are great tools to study the cell-cell interactions mediated by genes of interest. The embryonic transparency of zebrafish at early developmental stages permits direct visualization of the morphogenesis of tissues and organs at the cellular level. Here, we demonstrate a protocol to generate chimeric retinas and brains in zebrafish embryos and to perform live imaging of the donor cells. The protocol covers the preparation of transplantation needles, the transplantation of GFP-expressing donor blastomeres to GFP-negative hosts, and the examination of donor cell behavior under live confocal microscopy. With slight modifications, this protocol can also be used to study the embryonic development of other tissues and organs in zebrafish. The advantages of using GFP to label donor cells are also discussed.

Protocol

Day 1. Preparation

1. Set up fish crosses

Set up pairwise crosses of zebrafish in the evening. To prevent random mating before the desired time, use dividers to separate the female and male fish in the mating cassettes. GFP-expressing transgenic fish are used as a source of donor blastomeres. The pt104 transgenic line (Zou et al., 2008), used here, expresses GFP ubiquitously under the control of the EF1 promoter. GFP expression allows visualization of donor cells by live imaging. Other transgenic fish lines that express suitable fluorescent proteins may be used for specific needs.

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Discussion

In this video, we demonstrate a method for blastomere transplantation to analyze the retinal and brain development in zebrafish. This protocol can also be used to analyze the development of other tissues and organs by simply releasing the donor blastomeres to corresponding locations in the host embryos. The labeling with GFP molecules yields a cleaner background than labeling with fluorescent dye-conjugated Dextran (Ho and Kane 1990). This is because the debris of Dextran dyes from dead donor cells lasts much longer than.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
5 ¾" Glass pipette	Fisher Scientific	13-678-30
Borosilicate Glass Capillary	World Precision Instruments, Inc.	1B100F-4
FluoroDish	World Precision Instruments, Inc.	FD35
Vertical Needle / pipette Puller	David Kopf Instruments	Model 720
Micro Grinder	Marishige	EG-400
Micro Forge	Marishige	MF-830
Manual Microsyringe Pump	World Precision Instruments, Inc.	MMP
Forceps	Fine Science Tools	11232-20
Hydrofluoric Acid	Fisher Scientific	A147-1
Penicillin- Streptomycin	GIBCO, by Life Technologies	15140-122
Agarose	Research Products International Corp.	9012-36-6
Low melting agarose	Fisher Scientific	BP165-25
E3 egg water: 5 mM NaCl 0.17 mM KCl 0.33 mM CaCl2 0.33 mM MgSO4 0.1% methylene blue
5 mM NaCl 0.17 mM KCl 0.33 mM CaCl2 0.33 mM MgSO4 0.1% methylene blue
5 mM NaCl 0.17 mM KCl 0.33 mM CaCl2 0.33 mM MgSO4 0.1% methylene blue