This protocol delineates a way to label and trace the fate of small groups of cells zebrafish embryos using UV-uncaging of caged fluorescein, followed by whole mount immunolabeling to amplify the signal from the uncaged fluorescein.
A central problem in developmental biology is to deduce the origin of the myriad cell types present in vertebrates as they arise from undifferentiated precursors. Researchers have employed various methods of lineage labeling, such as DiI labeling1 and pressure injection of traceable enzymes2 to ascertain cell fate at later stages of development in model systems. The first fate maps in zebrafish (Danio rerio) were assembled by iontophoretic injection of fluorescent dyes, such as rhodamine dextran, into single cells in discrete regions of the embryo and tracing the labeled cell’s fate over time3-5. While effective, these methods are technically demanding and require specialized equipment not commonly found in zebrafish labs. Recently, photoconvertable fluorescent proteins, such as Eos and Kaede, which irreversibly switch from green to red fluorescence when exposed to ultraviolet light, are seeing increased use in zebrafish6-8. The optical clarity of the zebrafish embryo and the relative ease of transgenesis have made these particularily attractive tools for lineage labeling and to observe the migration of cells in vivo7. Despite their utility, these proteins have some disadvantages compared to dye-mediated lineage labeling methods. The most crucial is the difficulty we have found in obtaining high 3-D resolution during photoconversion of these proteins. In this light, perhaps the best combination of resolution and ease of use for lineage labeling in zebrafish makes use of caged fluorescein dextran, a fluorescent dye that is bound to a quenching group that masks its fluorescence9. The dye can then be “uncaged” (released from the quenching group) within a specific cell using UV light from a laser or mercury lamp, allowing visualization of its fluorescence or immunodetection. Unlike iontophoretic methods, caged fluorescein can be injected with standard injection apparatuses and uncaged with an epifluorescence microscope equipped with a pinhole10. In addition, antibodies against fluorescein detect only the uncaged form, and the epitope survives fixation well11. Finally, caged fluorescein can be activated with very high 3-D resolution, especially if two-photon microscopy is employed 12,13. This protocol describes a method of lineage labeling by caged fluorescein and laser uncaging. Subsequenctly, uncaged fluorescein is detected simultaneously with other epitopes such as GFP by labeling with antibodies.
1. Synthesis of Caged Fluorescein Dextran
2. Injection of Caged Fluorescein Dextran Into Zebrafish Embryos
3. Laser Uncaging of Fluorescein Dextran
4. Antibody Labeling and Detection of Uncaged Fluorescein Dextran
5. Representative Results:
After immunolabeling, there should be an enriched area of staining in the cells that were uncaged (Figure.1). It is not unusual to see a relatively high signal to noise ratio in >48 hour old zebrafish, due to spontaneous uncaging of the fluorescein.
Figure 1. Lineage labeling of zebrafish pineal complex. At 24 hpf, a portion of the anterior pineal anlage, indicated by foxd3:GFP expression, was uncaged using UV laser pulses. A) By 48hpf, a left-sided cluster of cells called the parapineal (red circle) has emerged from the uncaged domain (white circle). B) Uncaged fluorescein signal is enriched in the parapineal (red circle) and the pineal organ (white circle).
As described, this protocol provides a relatively quick lineage labeling method in zebrafish that is built upon the commonly used techniques of microinjection, microscopy, and immunofluorescence. We have found that laser uncaging to be the most efficient and cost effective way to uncage fluorescein in a localized fashion. This method could be used to lineage label with experimental endpoints as late as 4 dpf. However, as cells divide, the caged-fluorescein concentration per cell eventually decreases beyond detectable levels. In addition, there have been reports of spontaneous, non-specific uncaging of fluorescein in zebrafish larvae 11. As such, detection of uncaged fluorescein is most effective in early larval stage (48-72 hpf) or earlier.
Our preferred uncaging method is by use of a pulsed nitrogen laser. These are commonly used for cell ablations experiments and are sufficiently accurate to uncage singe cells or small groups of cells11-12. However, uncaging can also be accomplished using a confocal microscope; two-photon confocal microscopes have been used to achieve very precise uncaging12. On the other end of the spectrum, if precision is not required then a laser is dispensable. An epifluorescent microscope equipped with a DAPI filter set can be used to uncage through a small pinhole11.
We have found that detection of uncaged fluorescein using a primary antibody and fluorescent secondary antibody that emits at a red or far-red wavelength (e.g. Alexa 633) best suits our needs. This allows us to distinguish uncaged fluorescein from GFP expressed by a transgene (Figure 1).
The authors have nothing to disclose.
We would like to thank Dan Carlin for help in making caged fluorescein. In addition we would like to thank the following funding sources: Vanderbilt University Medical School Program for Developmental Biology Training Grant to JAC, and NIH HD054534to JTG.
Material Name | Type | Company | Catalogue Number | Comment |
---|---|---|---|---|
CMNB-caged fluorescein SE | Invitrogen | C20050 | ||
Aminodextran, 10kDa | Invitrogen | D1860 | ||
Zeba desalt spin columns | Pierce | 89889 | ||
goat anti-fluorescein antibody | Invitrogen | A11095 | ||
Alexa Fluor 633 Donkey anti-goat antibody | Invitrogen | A21082 | ||
35mmx10mm petri dishes | Becton-Dickinson | 351008 | ||
Upright compound microscope with 40x water immersion objective | Leica | DM6000B | ||
MicroPoint Manual Laser Illumination System | Photonic Instruments | |||
Phenylthiourea (PTU) | Alfa Aesar | L06690 | ||
Tricaine | Sigma | E10521 | ||
Proteinase K | Roche | 03115836991 | ||
Plastic thread | Any sewing supply store | |||
Agarose | Research Products International | A20090 | ||
SpeedVac | Thermo Scientific | Savant SPD131DDA | ||
Vortexing mixer | VWR | Vortex Genie 2 | ||
Pressure injector | Applied Scientific Instrumentation | MPPI-3 |