Tracking Cells in GFP-transgenic Zebrafish Using the Photoconvertible PSmOrange System

Summary

We established the photoconvertible PSmOrange system as a powerful, straight-forward and cost inexpensive tool for in vivo cell tracking in GFP transgenic backgrounds. This protocol describes its application in the zebrafish model system.

Abstract

The rapid development of transparent zebrafish embryos (Danio rerio) in combination with fluorescent labelings of cells and tissues allows visualizing developmental processes as they happen in the living animal. Cells of interest can be labeled by using a tissue specific promoter to drive the expression of a fluorescent protein (FP) for the generation of transgenic lines. Using fluorescent photoconvertible proteins for this purpose additionally allows to precisely follow defined structures within the expression domain. Illuminating the protein in the region of interest, changes its emission spectrum and highlights a particular cell or cell cluster leaving other transgenic cells in their original color. A major limitation is the lack of known promoters for a large number of tissues in the zebrafish. Conversely, gene- and enhancer trap screens have generated enormous transgenic resources discretely labeling literally all embryonic structures mostly with GFP or to a lesser extend red or yellow FPs. An approach to follow defined structures in such transgenic backgrounds would be to additionally introduce a ubiquitous photoconvertible protein, which could be converted in the cell(s) of interest. However, the photoconvertible proteins available involve a green and/or less frequently a red emission state¹ and can therefore often not be used to track cells in the FP-background of existing transgenic lines. To circumvent this problem, we have established the PSmOrange system for the zebrafish^2,3. Simple microinjection of synthetic mRNA encoding a nuclear form of this protein labels all cell nuclei with orange/red fluorescence. Upon targeted photoconversion of the protein, it switches its emission spectrum to far red. The quantum efficiency and stability of the protein makes PSmOrange a superb cell-tracking tool for zebrafish and possibly other teleost species.

Introduction

Exponentially improving imaging techniques allow following developmental processes over time periods of up to about four consecutive days³. In zebrafish and many other animal model systems, specific cells, tissues, axonal or vascular structures are marked by transgenic green or sometimes red or yellow fluorescent proteins to facilitate visualization. However, in most transgenic lines the transgene is not specifically expressed in the cells of interest but also additional structures, which hinders the precise tracking of for instance single cells or groups of cells.

Fluorescent photoconvertible proteins are well suited for cell tr....

Protocol

1. H2B-PSmOrange mRNA In Vitro Transcription and mRNA Purification

1. Linearize the H2B-PSmOrange containing pCS2+ plasmid using the NotI restriction enzyme according to manufacturer's instructions.
   Note: Perform the next steps using appropriate protections such as gloves and lab coat to prevent mRNA contamination and degradation.
2. Purify the linearized DNA using a PCR Purification kit or phenol-chloroform based methods according to manufacturers' protocols.
3. Use 1 µg of linearized template DNA for Sp6-mRNA transcription according to manufacturer's instructions.
4. Remove DNA by adding 1.0 U RNase free....

Results

Figure 1 illustrates an example of the PSmOrange photoconversion system. The pineal complex is a conserved structure in the vertebrate dorsal diencephalon. Like in many other vertebrates, this complex consists of the pineal organ in the center of the diencephalon and the left-sided parapineal cells. Elegant but time-consuming uncaging experiments showed that parapineal cells originate in the anterior part of the pineal organ¹³. In tg(foxD3:GFP); tg(flh:GFP) tra.......

Discussion

Transgenic embryos carrying fluorescent reporters have helped fundamentally to understand embryonic development. However, there is still the essential need for promoters to facilitate the specific visualization of particular structures. In their absence, researchers rely on techniques such as the photoconversion of fluorescent proteins to find out about the origin and development of their structure of interest. This in turn is a crucial prerequisite to identify the molecular mechanisms involved in its development. Techni.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
PCR Purification Kit	Qiagen	28104
mMESSAGE mMACHINE SP6 Transcription kit	Ambion	AM1340
RNeasy MiniElute Cleanup kit	Qiagen	74204
Plastic Pasteur	alpha laboratories	LW4000
Original H2B-PSmOrange Plasmid	Addgene	31920	The plasmid described in the paper is available in the Carl lab
FemtoJet Microinjector	Eppendorf	5247 000.013
Forceps (5 Inox)	NeoLab	2-1633
Lab-Tek II Chambered #1.5 German Coverglass System	Nunc	155382
Nikon A1R+	Nikon GmbH Germany	No Number
Nikon PLAN Apo λ 20X air objective	Nikon GmbH Germany	No Number
NIS Elements AR Software (v. 4.30.02)	Nikon GmbH Germany/Laboratory Imaging	No Number