Lineage Labeling of Zebrafish Cells with Laser Uncagable Fluorescein Dextran

Abstract

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 labeling 1 and pressure injection of traceable enzymes 2 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 time [3][4][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 zebrafish [6][7][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 vivo 7 . 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 fluorescence 9 . 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 pinhole 10 . In addition, antibodies against fluorescein detect only the uncaged form, and the epitope survives fixation well 11 . 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.

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“…For testing of our chemical decaging strategy in zebrafish embryos, the rhodamine sensor was conjugated to an amine-modified dextran polymer via NHS-activation of the carboxylate functionality on the pendant aryl ring, generating 10b . Dextran-conjugated fluorophores have a long history of use in zebrafish embryos, − as they are nontoxic, hydrophilic, and prevent diffusion out of the blastomeres. , …”

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“…Caged fluorescein dextran was synthesized as described (51). One-cell stage embryos were injected with 1 nl of RNase-free water containing Rad21 MO or a water control, and caged fluorescein dextran stock diluted 1:5.…”

Fgf signaling governs cell fate in the zebrafish pineal complex

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