Cell-Lineage Tracing in Zebrafish Embryos with an Expanded Genetic Code

Abstract

Cell-lineage tracing is used to study embryo development and stem-cell differentiation as well as to document tumor cell heterogeneity. Cre recombinase-mediated cell labeling is the preferred approach; however, its utility is restricted by when and where DNA recombination takes place. We generated a photoactivatable Cre recombinase by replacing a critical residue in its active site with a photocaged lysine derivative through genetic code expansion in zebrafish embryos. This allows high spatiotemporal control of DNA recombination by using 405 nm irradiation. Importantly, no background activity is seen before irradiation, and, after light-triggered removal of the caging group, Cre recombinase activity is restored. We demonstrate the utility of this tool as a cell-lineage tracer through its activation in different regions and at different time points in the early embryo. Direct control of Cre recombinase by light will allow more precise DNA recombination, thereby enabling more nuanced studies of metazoan development and disease.

Keywords: DNA recombination; lineage tracing; optogenetics; unnatural amino acids; zebrafish.

Figure 1

A) Structure of HCK. B) Active site of Cre recombinase showing replacement of K201 with HCK. Irradiation with 405 nm light releases the photocage to reveal the native lysine. C) Schematic of the DNA recombination reporter in the zebrafish line. Cre recombinase-catalyzed, site-specific DNA recombination switches cells from GFP expression to mCherry expression. D) Experimental design showing injection of RNA to express caged Cre recombinase, followed by localized irradiation with 405 nm light at the 6 or 14 hpf stage, and fluorescent imaging at 48 hpf.

Figure 2

Initial optimization of caged Cre recombinase photoactivation. A) Micrographs of mCherry fluorescence after irradiation with 405 nm light at 6 hpf. Confocal irradiation consisted of irradiation at ten z-planes with 1.6 or 3.1 μs pixel dwell times, whereas the LED irradiation lasted 5 min. Images were taken at 48 hpf. The three embryos with the highest level of mCherry expression were selected from each condition and pooled for fluorescence quantification and genomic DNA isolation for qPCR experiments. B) Quantification of mCherry fluorescence intensity by using ImageJ software. Error bars represent standard deviation of the three selected embryos. C) qPCR quantification of the amplified recombined gene relative to wild-type Cre recombinase. Error bars represent standard error of the mean from technical qPCR triplicates.

Figure 3

From left to right: micrographs of 48 hpf embryos that were not injected but irradiated with 405 nm LED for 5 min at 6 hpf (N =9), embryos expressing wild-type Cre recombinase (N =3), embryos expressing caged Cre recombinase in the absence of irradiation (N =5), and embryos expressing caged Cre recombinase after 405 nm LED irradiation for 5 min at 6 hpf (N =9).

Figure 4

Localized activation of Cre recombinase. Embryos expressing caged Cre recombinase were irradiated at 6 or 14 hpf in the areas highlighted in purple with the region expected to express mCherry highlighted in red on the 48 hpf embryo cartoon. Fluorescence images at 48 hpf show regional expression of mCherry that matches the zebrafish developmental plan, thereby demonstrating selective (spatially and temporally controlled) activation of Cre recombinase. mCherry expression was merged with bright-field images to show the localized region of Cre recombinase activation.