Cell Tracking Using Photoconvertible Proteins During Zebrafish Development

Summary

Here, we present a method for the photoactivated switch of photoconvertible fluorescent proteins (PCFPs) in the living zebrafish embryo and further tracking of photoconverted protein at specific time points during development. This methodology allows monitoring of cell biological events underlying different developmental processes in a live vertebrate organism.

Abstract

Embryogenesis is a dynamic process that is best studied by using techniques that allow the documentation of developmental changes in vivo. The use of genetically-encoded fluorescent proteins has proven a valuable strategy for elucidating dynamic morphogenetic processes as they occur in the intact organism. During the past decade, the development of photoactivatable and photoconvertible fluorescent proteins has opened the possibility to investigate the fate of discrete subpopulations of tagged proteins¹. Unlike photoactivatable proteins, photoconvertible fluorescent proteins (PCFPs) are readily tracked and imaged in their native emission state prior to photoconversion, making it easier to identify and select regions by optical inspection. PCFPs, such as Kaede², KikGR³, Dendra⁴ and EosFP⁵, can be shifted from green to red upon exposure to UV or blue light due to a His-Tyr-Gly tripeptide sequence which forms a green chromophore that can be photoconverted to a red one by a light-catalyzed β-elimination and subsequent extension of a π-conjugated system³. PCFPs and their monomeric variants are useful tools for tracking cells^6-10 and studying protein dynamics^11-14, respectively. During recent years, PCFPs have been expressed in different animal model, such as zebrafish⁶, chicken^7,8 and mouse^9,10 for cell fate tracking. Here we report a protocol for cell-specific photoconversion of PCFPs in the living zebrafish embryo and further tracking of photoconverted proteins at later developmental stages. This methodology allows studying, in a tissue-specific manner, cell biological events underlying morphogenesis in the zebrafish animal model.

Protocol

1. Obtaining Embryos for the Photoconversion Assay

Upon in-crossing of transgenic zebrafish expressing a PCFP the brightest which are homozygote embryos are chosen, and grown at 28.5 °C until the desired embryonic stage¹⁵.

Note 1: This preselection will allow scanning embryos with low laser power thereby preventing phototoxicity and photobleaching.

Note 2: We suggest to use low-intensity light when visualizing embryos and to maintain them in the dark conditions due to high sensitivity of PCFPs, such as Kaede and KikGR, to light exposure including daylight.

Discussion

In recent years, transgenic animal models for the expression of photoconvertible proteins such as Kaede or KikGR have been generated. These animals develop normally, indicating that these proteins have no toxic effects on embryonic development. The first report about a photoconversion assay for tracking cells in animal embryos was performed by Hatta and collaborators by injection of mRNA or DNA encoding Kaede into one-cell zebrafish embryos for ubiquitous expression, or by expression of Kaede and KikGR in neural tissue b.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Materials:
Necessary equipment for raising fish and collecting eggs (see the Zebrafish Book22 for details).
Stereomicroscope	Leica	Lecia MZ6 Stereomicroscope	Equipped with laser operating at 488 nm
Confocal laser scanning microscope	Zeiss	Zeiss LSM 710 NLO	equipped with lasers operating at 488 nm, 561 nm, and 405 nm
Heating block
Pair of Dumont #5 forceps
Cover-glass-bottomed culture dish	MatTek Corp		Ashland MA USA
Microloader Pipette Tips	Eppendorf
Vortex
needles
Reagents:
Egg water medium
60 μg/ml Instant Ocean Sea Salts in ddH2O
E3 medium
5 mM NaCl
0.17 mM KCl
0.33 mM CaCl2
0.33 mM MgSO4
0.00001% (w/v) Methylene Blue
4 mg/ml Tricaine stock solution
400 mg Tricaine (3-amino benzoic acidethylester)	Sigma-Aldrich
97.9 ml ddH2O
2.1 ml 1M Tris (pH9)
Adjust at pH 7, aliquot and store -20 °C
1% LMA (Low melting temperature agarose, Lonza)
1g LMA/100 ml E3 medium 1X
Aliquot and store at 4 °C